Cervical Cancer HeLa Cell Autocrine Apoptosis Induced by Co

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Cervical Cancer HeLa Cell Autocrine Apoptosis Induced by Co-immobilized TNF-# plus IFN-# Biomaterials Jian Li, Yuxiao Zhang, Liyi Chen, Xinhua Lu, Zhibin Li, Yongyong Xue, and Yan-Qing Guan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18277 • Publication Date (Web): 13 Feb 2018 Downloaded from http://pubs.acs.org on February 13, 2018

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ACS Applied Materials & Interfaces

Cervical Cancer HeLa Cell Autocrine Apoptosis Induced by Co-immobilized TNF-α plus IFN-γ Biomaterials

Jian Li†,‡, Yuxiao Zhang§, Liyi Chen§, Xinhua Lu§, Zhibin Li§, Yongyong Xue†,‡ Yan-Qing Guan*,†,‡,§

†

MOE Key Laboratory of Laser Life Science &Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China.

‡

Joint Laboratory of Laser Oncology with Cancer Center of Sun Yet-sen University,South China Normal University, Guangzhou 510631, China. §

School of Life Science, South China Normal University, Guangzhou 510631, China.

KEYWORDS: Co-immobilized IFN-γ plus TNF-α, Cervical cancer, TNF-α, IFN-α, Autocrine Signaling

*Corresponding author at: School of Life Science, South China Normal University, Guangzhou 510631, P. R. China. Tel.: (+86-20)85211241; E-mail address: [email protected] (Y. Q. Guan)

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ABSTRACT: Using the external methods to induce the death of cancer cells is recognized as one of the main strategies for cancer treatment. Research indicated that TNF-α is frequently used in tumor biotherapy while IFN-γ can directly inhibit tumor cell proliferation. In our study, the TNF-α and IFN-γ were co-immobilized on Polystyrene material (PSt) or Fe3O4-Oleic acid nanoparticles (NPs). Then the structural changing of these two proteins can be observed. Meanwhile, the expressions of both TNF-α and IFN-α increased significantly, as determined by gene microarray analysis, however, in the presence of TNF-α plus IFN-α inhibitors, TNF-α and IFN-α did not increase in HeLa cells induced by co-immobilized TNF-γ plus IFN-α. our results indicate that such changing can stimilate HeLa cells to autocrine TNF-α and IFN-α, by which the apoptosis of HeLa cells could be further induced. It is for the first time that the apoptosis of HeLa cells being induced by the autocrine is reported in our study. In addition, we performed Elisa, RT-PCR, flow cytometry, and western blot analyses, as well as a series of analytical tests at the animal level. our data also indicate that the co-immobilized IFN-γ plus TNF-α NPs has apparent effects for cancer treatment in vivo, which is great significance to translate into clinical medicine.

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1. INTRODUCTION Cervical cancer is the third most common malignancy in women worldwide, behind breast and colorectal cancers. This is particularly serious in developing countries, where its incidence is just under that of breast cancer.1 There are an estimated 50 million new cases of cervical cancer and more than 24 million deaths each year around the world, and about 1 / 5 of the total new cases occur in China. Moreover, this cancer has recently been occurring in younger populations. Therefore, the prevention and treatment of cervical cancer are regarded as an important focus of scientific research. IFN-α, a type of cytokine, has been used for over 30 years in clinical applications, having been proven to have positive therapeutic effects on human malignant tumors, such as hairy cell leukemia, lymphoma, renal cell carcinoma, malignant melanoma, basal cell carcinoma and squamous cell carcinoma. An impressive body of literature indicates that its antitumor mechanism is composed of two aspects. One is direct toxicity to tumor cells and the other is indirect inhibition by immune regulation. Similarly, TNF (usually referring to TNF-α) is a cytokine secreted by immune cells with the ability to inhibit tumor cell growth and induce degenerative changes in tumors.2-18 Recently, there have been many reports about interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) and their respective actions on tumor cells, as well as their antitumor effects when combined with other drugs.19 Nevertheless, there are very few reports about the synergistic effect of IFN-γ and TNF-α, although some studies have been published on their synergistic effect in other diseases.20-25 In earlier research, we recorded two death mechanisms. The first was the IFN death receptor pathway and the other was like-programmed cell death. Cell death and apoptosis did not occur through the secretion of traditional caspase related proteins, instead were rather caused by EndoG released by the mitochondria.26,27 However, in this study, we found a new death mechanism. Cytokine autocrine phenomenon refers to the ones that the cells secret cytokines and then such cytokines act on the secretory cells themselves to regulate their own function of a process. Paracrine is another phenemenon that the hormones or regulatory factors play a regulatory role through the cell gaps with neighboring cells.28,29 In recent years, many 3



substances can be found in a variety of tumor cell culture media, and such substances have significant inhibition for a variety of primary cells or cell proliferation. They frequently play an important regulatory role through receptors or cytokines in autocrine or paracrine manner.29,30 Therefore, it is a more common phenomenon that autocrine factor can inhibit tumor cells from growing. TNF-α was initially considered to only be secreted by macrophages, but more recent research has shown that a wide variety of tumor cells can secrete TNF-α and other cytokines, including adult T-cell and B cell lymphomas, chronic and acute lymphoblastic leukemia, and cervical epithelial ovarian, breast, ovarian, colon, pancreatic, and lung cancers. In this study, we focused on both IFN-γ and IFN-α. TNF-α and IFN-α can be spontaneously synthesized and secreted by HeLa cells, and both can specifically bind their receptors, which activates signal transduction pathways and promote the apoptosis of cervical cancer cells. After induction by TNF-α and IFN-γ, Hela cells secrete more different level of TNF-α and IFN-α, thereby promoting increased apoptosis. However, in the meantime TNF-α and IFN-γ still act on both cancer and normal cells, resulting in severe side effects and causing many patients to discontinue intravenous use. There are doubt whether methods can be discovered to reduce the side effects while maintaining the original treatment efficacy. Therefore, this research is based on the combination of IFN-γ and TNF-α, we investigated their joint action on cervical cancer cells, and their mechanisms of cell death. Light grafting method was used to fix the two drugs on a PSt board, in order to prepare the optically activated drug.

2. EXPERIMENTAL SECTION 2.1. Chemicals. IFN-γ, TNF-α, lauryl sodium sulfate and N-(4-diAzPho) aniline hydrochloride were purchased from Shanghai clone biotechnology co. LTD, China. Anhydrous ethanol, dimethyl sulfoxide (DMSO), gallic acid, Dimethylaminopyridine (DMAP), Dicyclohexylcarbodiimide (DCC), 1-(3-dimethylamino propyl)-3-ethylcarbonimide (EDC) and N-hydroxy succinimide (NHS) were purchased from Sigma Co. Ltd, USA. Recombinant human insulin, phosphate buffer saline (PBS), PEG6000 and Fluorescein 4


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isothiocyanate (FITC) labeled insulin were purchased from Guangzhou Weijia biological technology Co. Ltd, China. Fetal bovine serum (FBS), DMEM (High Glucose) were purchased from Gibco life technologies Co. Ltd, USA.

2.2. Synthesis of Biomaterials. 50 µg of IFN-γ and TNF-α was dissolved in an AzPhidoaniline hydrochloride dimethylamide solution respectively at a concentration of 7.7 × 10-2 mg / mL and the activity of IFN-γ was measured by magnetic stirring at 48 h for 4 ℃ (ice bath, in the dark). The photocatalytic TNF-α and the photocatalytic IFN-γ were added to a 24-well cell culture polyethylene plate (PSt) at a concentration of 10 ng / mL each. The photoactive TNF-α and IFN-γ were mixed and homogenized with a pipette, so that the mix evenly covered the plate surface, at 4 ℃ in the dark. They were then exposed to UV lamp (15 W) under 2 cm irradiation for 10 min, and AzPhide instability and other polymers were used to form chemical bonds, immobilizing the photoactive IFN-γ and TNF-α onto the PSt material.27,31

2.3. Synthesis of Fe3O4-Oleic Acid. Fe3O4-Oleic Acid were synthesized in our laboratory, following the procedure reported in literature.32

2.4. Preparation of Nano-drug. Photoactive TNF-α (10 µg), photoactive IFN-γ (10 µg) and Fe3O4-Oleic Acid magnetic nanoparticles (NP, 1 µg) were added to 10 mL PBS. After 48 h reaction at 4 ℃ (in the dark), the solution is ultrasound for 30 min, and dispersed with a ultrasonic cleaner for a period of time after the transfer to the Petri dish. The solution is placed shaker, 100 rpm / min, while using UV lamp (125 W) above the liquid surface 10 cm irradiation. The use of AzPhido is very lively. Then photoactive TNF-α and IFN-γ was co-immobilized on Fe3O4-Oleic Acid magnetic nanoparticles (NP).

2.5. Cell Culture and Treatment. HeLa cells obtained from Sun Yat-Sen University, and were grow in RPMI 1640 medium (Gibco BRL) with 10 % FBS, supplemented with 0.03 µg / mL penicillin and 0.05 µg / mL streptomycin in a humidified 5 % CO2 atmosphere at 37 ℃. First, the cells were seeded at 1 × 105 cells / mL into a 24-well polystyrene culture plate 5



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with serum-free medium and were induced by adding PSt-co-immobilized TNF-α plus IFN-γ to a final concentration of 10 ng / well. After stimulation by the PSt-co-immobilized TNF-α plus IFN-γ for 12 or 24 h, respectively, these cells were washed three times using phosphate-buffer saline (PBS), harvested, and then used for other in vitro experiments.

2.6. Gene Chip. We collected two samples, the free and the co-immobilized groups. The free group was induced by free IFN-γ and TNF-α and the co-immobilized group was induced by PSt-co-immobilized IFN-γ plus TNF-α. Each was collected after 24 h of culture on the 24-well PSt. We sent our samples to CapitalBio Corporation in Beijing for analysis.

2.7. Real-Time Fluorescent Quantitative PCR for IFN-α and TNF-α Mrna. RNA was collected from HeLa cells induced in different ways, named the control which was only the cells, the free group, which was induced by PSt + AzPhIFN-γ plus AzPhTNF-α, and the co-immobilized group, which was induced by PSt-co-immobilized IFN-γ plus TNF-α. Each was collected after culturing for 12 or 24 hours on the 24-well PSt. Reverse transcription was performed with the TaKaRa PrimeScript TMRT reagent kit(Prefect Real Time). PCR was used to

detect

IFN-α

and

TNF-α

using

the

following

primers:

IFN-a:

Forward:

5’-ACCTTTGCTTTACTGGTGGCC-3’, Reverse: 5’-TTGTCTAGGAGGGTCTCATCCC-3’; TNF-α: Forward: 5’-GTGCTCCTCACCCACACCAT-3’, Reverse: 5’-GCAAAGTCGAGAT AGTCGGGC-3’.

2.8. Inhibition both of TNF-a and IFN-γ. Similarly, the HeLa cells (1 × 105 / well) were seeded onto the 24-well cell culture polystyrene plates and treated respectively with PSt + AzPhIFN-γ plus AzPhTNF-α or PSt-co-immobilized TNF-α plus IFN-γ for 12 h and 24 h. The cells were then collected and washed with Dulbecco PBS, re-suspended in binding buffer, and incubated with Annexin V-FITC for 15 min at room temperature in the dark. After the centrifugation, Annexin V-FITC was removed and the HeLa cells were stained with PI in binding buffer. Finally, the HeLa cells were immediately analyzed with flow cytometry (Becton Dickinson, FACSC Alibur, San Jose, CA) using the Cell Quest Program. 6


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2.9. Statistical Analysis. All data were subjected to mixed model analysis of variance, and the SPSS software was used to compare the differences in various experimental groups at each sampling day. The data were expressed as means ± S.E.M and p < 0.05 was considered to be a statistically significant difference. For further details, refer to the Supporting Data

3. RESULTS 3.1. Synthesis of PSt-Co-immobilized IFN-γ plus TNF-α. The preliminary results showed that TNF-α and IFN-α can induce autologous cell death in the form of PSt-co-immobilized IFN-γ plus TNF-α biomolecules, and that can react to self-cancer cells, thereby enhancing cell death. PSt + AzPhIFN-γ plus AzPhTNF-α, however, induced HeLa cell death, but the effect of free cytokine compared to co-immobilized cytokines was significantly lower than that of the co-immobilized biomaterials. We then investigated PSt-co-immobilized IFN-γ plus TNF-α after UV irradiation and found that the tertiary structure of the two cytokines interacted with each other and caused changes in the structures of the two proteins (Figure 1). This structural change not only alleviated the side effects caused by individual administration, but also promoted the apoptosis of tumor cells.

3.2. FRET Measurement in Cells with Varying Donor or Acceptor Levels. In order to verify these results after uv graft to the PSt, and that the structures of these two proteins changed, we used the FRET technique. Figure 2c shows that the as a donor of TNF-α fluorescence intensity is much lower than when it exists alone (fluorescence quenching), and the launch fluorescence of IFN-γ as a receptor is greatly enhanced (sensitized fluorescence). This proves the protein macromolecular conformation changes and demonstrates that the distance between the two molecules was within the 10 nm range.

3.3. Analysis of X-ray Diffraction and Contact Angle Results. We used x-ray diffraction to analyze the structure of IFN-γ and TNF-α after their UV-induced co-immobilization on PSt. As shown in Figure 2b, compared with the IFN-γ plus TNF-α graft 7



on PSt after photo-activation, in the vicinity of 45.5°, the IFN-γ plus TNF-α graft on PSt presented with a diffraction peak. In addition, there were differences of about 100 and 200, showing variation in the two crystal forms. Moreover, we also tested the grafting of free AzPhIFN-γ and AzPhTNF-α to PSt. The resulting comparison showed several diffraction peak differences. In the vicinity of 45.431°, the free AzPhIFN-γ graft on PSt displayed a diffraction peak, and the peak in the vicinity of 100 also showed that the diffraction peak was different. In addition, in the vicinity of 30.774°, the free AzPhIFN-γ graft on PSt displayed a very obvious diffraction peak, and there were three different diffraction peaks at about 00, 200, and 310. In conclusion, after the comparison of several kind of materials, we found that when IFN-γ and TNF-α were co-immobilized on PSt, their crystal forms show differences compared to free IFN-γ and TNF-α grafted onto PSt. Subsequently, we measured the wettability of the sample with contact angle meter. As shown in Figure 2a, the angles of PSt-Immobilized-TNF-α group, PSt-Immobilized-IFN-γ group and PSt + AzPhIFN-γ plus AzPhTNF-α group are similar. Left and right’s angles are below 45°, which suggests that the three kinds of materials bear the great potential of hydrophilic. The same hydrophilic can prove that the structure of single grafting material has not changed. However, the left and right angle of PSt-Co-immobilized IFN-γ plusTNF-α are 77.1° and 75.8° respectively, which suggests PSt-Co-immobilized IFN-γ plus TNF-α has weaker hydrophilicity. According to such hydrophilic changes, we can conclude that the structure of PSt-Co-immobilized IFN-γ plus TNF-α has changed by the ultraviolet light irradiation.

3.4. The Characterization of Nano-drugs. Three characterization tests were performed on the nano-drugs. Four kinds of NPS are Fe3O4-OA-Immobilized-TNF-α, Fe3O4-OAImmobilized-IFN-γ, Fe3O4-OA + AzPhIFN-γ plus AzPhTNF-α and Fe3O4-OA-Coimmobilized IFN-γ plus TNF-α respectively. We first examined the morphology and size of the nano-drugs (Figure 3c). SEM results show that the shape of four nano-drugs are nearly spherical, and evenly distributed. Particle size detection showed that the size of four nano-drugs is 140 nm, 110 nm, 78 nm, 300 nm, respectively (Figure 3b). According to SEM 8


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morphology and particle size changes, we can be initially judged that TNF-α and IFN-γ have been successfully grafted to the Fe3O4-OA. Subsequently, we use Fourier transform infrared spectroscopy (FTIR) to solve that the change of chemical bonds during grafting. FTIR data for the four types of nano-drugs are summarized in Figure 3a. None of the four nano-drugs showed the peak of -N3 indicating that all of the substances were attached to Fe3O4-OA. Fe3O4-OA + AzPhIFN-γ plus AzPhTNF-α and Fe3O4-OA-Co-immobilized IFN-γ plus TNF-α appear peaks at 2851.6 cm-1, where the chemical bond represented for C-N. Due to the Fe3O4-OA + AzPhIFN-γ plus AzPhTNF-α is exposed to the faint daylight in operation process, so the peak of C-N also appear in the Fe3O4-OA + AzPhIFN-γ plus AzPhTNF-α group. In contrast, after UV irradiation, the peak at Fe3O4-OA-Co-immobilized IFN-γ plus TNF-α is more pronounced. This kind of change can show that the two protein factors are linked together after UV irradiation, which made C-N increase. In summary, Fe3O4-OA-Co-immobilized IFN-γ plus TNF-α was synthesized successfully.

3.5. Microarray Analysis of HeLa Cells for Genes Associated with TNF-α and IFN-γ. In order to study the effects of the different biomaterials on HeLa cells, we obtained a gene expression profile chip to analyze the expression status of genes in HeLa cells induced by either PSt-co-immobilized IFN-γ plus TNF-α or free IFN-γ and TNF-α. The results showed that the expression of IFN-α was up-regulated by 7.9383 times in the co-immobilized group compared with free, and the expression of TNF was over-expressed by 3.4406 times in HeLa cells treated with the co-immobilized biomaterials. In addition, the expressions of other genes associated with IFN and TNF also changed to different degrees. All of the above results are displayed in Figures 4 and 5. In light of these findings, we put forward a hypothesis that an upregulated secretion of TNF-α and IFN-α was induced by the co-immobilized group compared with that of the free and control groups, and that IFN-α and TNF-α might bind to their receptors and trigger related pathways in HeLa cells, inducing cells death.

3.6. The mRNA Level of IFN-α and TNF-α. Next, in order to further develop our hypothesis, we used real-time quantitative RT-PCR to detect the relative expression of TNF-α 9



and IFN-α mRNA. According to the Elisa results, seen in Figure 6b, the relative mRNA level of IFN-α after a 12 h induction were the highest in the control (CK) group, followed by the free (F) and the co-immobilized (C) groups. However, the 24 h results were quite different, in which the C group showed the highest expression levels, followed by the control and F groups. The TNF-α results were similar to those of IFN-α, which were consistent with the Elisa results. All the results driven from this study show that when the HeLa cells were induced by PSt-co-immobilized IFN-γ plus TNF-α, the expression of IFN-α and TNF-α both increased obviously.

3.7. Protein Expression and Localization of TNF-α and IFN-α. Moreover, in order to further analyze the effects of the graft material on cell secretion, we used confocal microscopy to observe the fluorescence of two factors every 4 hours from 0 to 48 hours. As seen in Figure 6c, in both factors, TNF-α and IFN-α, the PL relative intensity increased with increasing time, with the results from 24 h showing the strongest fluorescence intensity. However, about 40 hours later, the fluorescence intensity had begun to show a decreasing trend. Together, these fluorescence results show that the expression of IFN-α and TNF-α both increase when induced by PSt-co-immobilized IFN-γ and TNF-α. Furthermore, this expression level was dependent on induction time.

3.8. Concentration Variation of IFN-α and TNF-α at Different Induction Times. We detected the secretions of TNF-α and IFN-α with a TNF-α Elisa and IFN-α Elisa kits at 6, 12, 18, 24, 72 and 144 h. The results are shown in Figure 6d, in which the secretion of IFN-α triggered by the PSt-co-immobilized IFN-γ and TNF-α were seen to be the highest compared with the CK and the F groups. This was particularly true at 24 h, at which the expression of IFN-α was 19.5 µg / L, while the CK and the F groups were 8.1 µg / L and 11.1 µg / L, respectively. Similarly to the expression of IFN-α, the expression trend of TNF-α also showed the C group to be the highest. At 24 h, the C group expression level was 264.2 ng / L, while the CK group and the F group were 149.7 ng / L and 185.5 ng / L, respectively. These results demonstrate that the expressions of IFN-α and TNF-α were up regulated when HeLa cells 10


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were treated with the PSt-co-immobilized IFN-γ and TNF-α. IFN-γ has been reported to up regulate the expression of type I IFNs and other IFN-γ target genes in tumor cells, leading to cell.22

3.9. Expression of TNF-α and IFN-α Detected by Western Blot. We used western blot analysis to detect the expressions of IFN-α and TNF-α in HeLa cells, in order to confirm the RT-PCR results, at 12 hours and 24 hours. When the HeLa cells had been induced 12 hours, the protein expressions of TNF-α and IFN-α were the lowest in the CK group, followed by the F group, with the highest expression in the C group. When the HeLa cells had been induced for 24 hours, the results were similar to the results of the 12-hour induction. Western blot can detect the protein expression of cells induced by drugs, so the results in Figure 7a, show the expression trends of IFN-α and TNF-α for HeLa cells induced by the C and F groups.

3.10. Expression of IFN-α and TNF-α both Decreased Secretion of Inhibition in HeLa Cells. Furthermore, we also verified the expression of IFN-α and TNF-α protein after inhibition. As we can see in Figure 7b, compared with the CK group (without inhibitors), we found that when the cells were induced by inhibitors, the expressions of IFN-α and TNF-α decreased after 24 h.

3.11. Mortality Rate Measured by Flow Cytometry. The above results prove that the two factors can promote cell apoptosis, but we then used the streaming and western blot analyses to detect changes in the rate of cell apoptosis after the addition of an inhibitor treatment, as well as the expression of the two protein factors. The Elisa and PCR experiments described above both supported our hypothesis, so we used TNF-α and IFN-α inhibitors to hinder the expressions of TNF-α and IFN-α, followed by flow cytometry to detect the cell mortality rate before and after inhibition. In order to verify the effects of the two factors on promoting apoptosis, we first collected six groups of HeLa cells: the control (CK), PSt + AzPhIFN-γ plus AzPhTNF-α (F), PSt-co-immobilized IFN-γ plus TNF-α (C), control with inhibitors, PSt + AzPhIFN-γ plus 11



AzPhTNF-α with inhibitors and PSt-co-immobilized IFN-γ plus TNF-α with inhibitors. We found that when the cells were induced for 24 hours, the cell mortality rate were 11.12 %, 10.72 %, 17.65 %, 10.40 %, 5.07 %, and 5.90 %, respectively (Figure 7c). We can see that the PSt-co-immobilized IFN-γ plus TNF-α biomaterials group had the highest cell death rates of almost 20 % when without IFN-α and TNF-α secretion inhibitors, the cell death rates of the control group and the F group were almost 10 %. The difference between the C group and the CK group was significant (***, P < 0.001). With the addition of IFN-α and TNF-α secretion inhibitors, there was a significant decrease in the C group cell death rate, only about 6 %, while the mortality in the CK group and the F group decreased not obvious, especially the CK group. Compared with the CK group, the C group showed a significant reduction in cell death (***, P < 0.001). Therefore, based on the results of cell counting and flow cytometry, we can conclude that: inhibition of IFN-α and TNF-α secretion inhibits the secretion of IFN-α and TNF-α induced by PSt-co-immobilized IFN-γ plus TNF-α biomaterials in HeLa cells, while HeLa cells autocrine IFN-α and TNF-α can cause HeLa cell death.

3.12. Determination of Cell Count by Flow Cytometry after Inhibition. In order to verify the apoptotic effects of the two factors, we treated the cells differently: no inhibitors, TNF and IFN-γ inhibitors were divided into control, PSt + AzPhIFN-γ plus AzPhTNF-α, and PSt-co-immobilized IFN-γ plus TNF-α groups. After 24 hours, we found that when the cells were induced for 24 hours, the cell mortality rates decreased and when the cells were exposed to inhibitors, the cell mortality rates decreased. In addition, at 24 hours, the cell mortality rate of the CK group was the highest compared with the other two groups, suggesting that the effect of the PSt-co-immobilized IFN-γ plus TNF-α on HeLa cells was delayed (Figure 7d).

3.13. Inhibiting Growth of a Cervical Tumor and Increasing Life Span in Mice. The in vitro assays suggested that cervical cancer in vivo may be inhibited by the nano-drugs. To verify this hypothesis, in vivo tumorigenesis assays were performed in nude mice with the transplantation of HeLa cells. The results showed that the nano-drugs treatment (10 ng / µL, 0.2 mL) significantly suppressed the growth of cervical tumors. At day 0, the tumor size of the three groups of nude mice was not significantly different, but with treatment, the tumor 12


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size of the three groups was significantly different by the 28 th day. The tumors in the CK and F groups were larger than those of C group. Due to drug inhibition, the tumor size was smaller, which proved that the nano-drugs negatively affected the tumor (Figure 8b). As shown in Figure 8d, the tumor volume only decreased in the C group, and the volume of the control tumors increased steeply in 28 days, resulting in only 20 % of normal saline treated mice (CK group) surviving after 28 days. The mice treated with nano-drugs showed prolonged survival. At the same time, the weights of nude mice were relatively stable in the nano-drugs group, while a significant decline was seen in the F and CK groups. Figure 8c shows the immunohistochemistry results of TNF-α and IFN-α protein expression in tumor tissue. The injection of nano-drugs into tumor tissue resulted in significantly enhanced fluorescence, revealing that both TNF-α and IFN-α proteins were upregulated, increasing autocrine signaling. This is consistent with the results at the cell level. As seen in Figure 8e, the immunoblotting results in nude mice were consistent with the experimental TNF-a and IFN-α protein expression results, induced by PSt-co-immobilized TNF-α plus IFN-γ modified medical polymer materials in HeLa cells. The protein expression also showed a tendency to increase, indicating that tumor cells could secrete their own TNF-α and IFN-α cytokines after drug treatment, thereby further promoting tumor cell apoptosis. These results were consistent with those of immunohistochemical staining.

3.14. Effect of Nano-drugs on Tumor Progression and Toxicity Data in Nude Mice. In order to further determine the distribution of nano-drugs in nude mice, the morphology of the excised tumors, hearts, livers, spleens, lungs, and kidneys were determined and are shown in Figure 8. The blue color for multi-target MNPs and the red color for tumor cell nuclei are evident. However, the Prussian blue staining of tumor tissue in the C group shows a more remarkable blue image than that of the F group, and is not seen at all in the CK group. Almost no multi-target MNPs could be found in other tissue. Moreover, we assessed the toxicity of the nano-drugs in nude mice by counting white blood cells (WBC), blood platelets (PLT), and red blood cells (RBC). There was no significant difference compared to the normal index, suggesting no toxic effects of the nano-drug in vivo (Figure 9). Thus, all these data indicate that the nano-drug can effectively inhibit tumor growth and reduce the mortality of 13



tumor-burdened nude mice, with no toxic effects.

4. DISCUSSION Cervical cancer is a serious malignancy in women worldwide.31,33 Cancer can weaken the role of autoimmune system and the common methods for cancer treatment are radiation therapy, chemotherapy, surgery, or a combination of these.34,35,36 If cervical cancer is caught and treated early, whether with surgery or radiation therapy, it is highly curable, but if found in the middle or late stages, treatment efficacy is poor.37 In nearly 30 years, the cure rate of patients with terminal cancer has hardly improved.38,39 TNF-α and IFN-α are two protein factors known to promote tumor cell apoptosis and to exert an anti-tumor effect.34,40-42 Our results suggest that cervical cancer HeLa cells can spontaneously secrete a small amount of TNF-α and IFN-α, and that treatment with IFN-a and TNF-γ can promote this autocrine phenomenon.43-44,45-49 Nanoparticle drugs and associated cancer treatment technologies have been receiving widespread attention,50 and magnetic nanoparticle drugs have matured in our preliminary works.32 The magnetic nanoparticle drugs have their physical therapy advantages over others,51,52 such as small particle size, large specific surface, high capacity coupling, and magnetic response.53 Therefore, in this study, magnetic nanoparticles were first decorated by the anti-cancer factors, then employed for in vivo experiments. Previous laboratory work has found that PSt-co-immobilized TNF-α plus IFN-γ induces a more pronounced cell death in HeLa cells.18,19 Furthermore, PSt-co-immobilized TNF-α plus IFN-γ did not enhance the presence of STAT1 in the JAK / STAT pathway.19 In fact, the effects of PSt + AzPhIFN-γ plus AzPhTNF-α induced death and signal transduction were significantly different than those of PSt-co-immobilized TNF-α plus IFN-γ in terms of death-related proteins. The morphological characteristics of PSt-co-immobilized IFN-γ plus TNF-α treated HeLa cells were also examined and PSt-co-immobilized IFN-γ plus TNF-α was found to induce death similar to apoptosis, including the formation of apoptotic bodies.19 We therefore found that PSt-co-immobilized IFN-γ plus TNF-α induced cervical cancer HeLa cell death apoptotic mechanisms, which is a type of death that has not been previously found 14


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in cervical cancer HeLa cells. Further exploration is necessary of the mechanisms by which PSt-co-immobilized TNF-α plus IFN-γ induces the apoptosis of cervical cancer HeLa cells and the exploration of the changes in protein conformation during the formation of co-immobilized TNF-α plus IFN-γ.54-56 In order to understand the PSt-co-immobilized TNF-α plus IFN-γ biomaterial structure, we carried out x-ray diffraction. Through that analysis we clearly determined that there was a large difference in the structure of co-immobilized TNF-α plus IFN-γ grafted to PSt and that of AzPhTNF-α and AzPhIFN-γ alone grafted to PSt. The FRET test showed that, after exposure to ultraviolet light, the structural distances between these two proteins were within 10 nm. Co-immobilized TNF-α plus IFN-γ grafting to PSt caused structural changes that inevitably led to changes in cervical cancer HeLa cells.57 We performed a gene microarray analysis and the results showed that, compared to the free and control groups, the secretion of TNF-α and IFN-α was significantly increased by exposure to the PSt-co-immobilized TNF-α plus IFN-γ group. This explains our synthesis of PSt-co-immobilized TNF-α plus IFN-γ causing TNF-α and IFN-α production by HeLa cells. Therefore, we boldly propose that IFN-α and TNF-α may associate with their receptors and trigger HeLa cell apoptosis related pathways, which is an autocrine-induced apoptosis or split long block, and which we have determined to be a third death mechanism in HeLa cells. There are many studies of the phenomenon of substances secreted by cancer cells that affect their living environment. For example, in 2012, researchers found that autocrine interleukin-35 in pancreatic cancer can promote PCAN cell proliferation and inhibit apoptosis.58 At the same time, National University of Singapore found that autocrine human growth hormone increased the tumor initiating capacity of negative breast cancer cells.59 For cancer cells, autocrine-induced apoptosis or split long block has already been reported. As early as 2007, the University of Texas found that autocrine TNF-α signaling in lung cancer cells can cause apoptosis.60 At the same time, the Si chuan University in China also determined the role of autocrine TNF-α in cancer cells during the induction of apoptosis.61 Researchers also discovered that autocrine IGF-1/AKT signaling in pancreatic cancer cells can lead to prolonged dormancy.62 Our findings are in agreement with those of these previous 15



studies. Although there have been reports that cancer cells can induce autologous apoptosis or long block by autocrine signaling,53,63 this mechanism had not been demonstrated for cervical cancer HeLa cells until this study, in which it is very clear that cervical cancer HeLa cells can send autocrine IFN-α and TNF-α signals to induce apoptosis or the long block phenomenon, which is a third death mechanism. We performed in vivo and in vitro assays on co-immobilized TNF-α plus IFN-γ and the results showed a significant increase in both IFN-α and TNF-α. The PCR results also showed a significant increase in IFN-α and TNF-α mRNA at 24 hours, and then IFN-α and TNF-α were measured for PSt-co-immobilized TNF-α plus IFN-γ in their ability to induce HeLa in the presence of TNF-α and IFN-α inhibitors. The PSt-co-immobilized TNF-α plus IFN-γ group showed a significant decrease, while the free group did not change. This result also confirms that the PSt-co-immobilization TNF-α plus IFN-γ causes an increase in TNF-α and IFN-α expression, leading to the occurrence of autocrine apoptosis in cervical cancer HeLa cells. At present, there have been no new breakthroughs in the research of the first two mechanisms of HeLa cell death.34 The findings of this study, therefore, that autocrine induced apoptosis or long block is the third death mechanism, have great practical significance. This is a new potential treatment for cervical cancer, caused by HeLa's own secretions, and may minimize the harm to the human body.

5. CONCLUSION Cervical cancer is one of the most common malignancies in the world. Our study found that when HeLa cells were induced by co-immobilized IFN-γ plus TNF-α, the expressions of both IFN-α and TNF-α increased obviously at all three levels (gene, mRNA, and protein). The emergence of this phenomenon proves two points. First, when IFN-γ and TNF-α are linked, their protein structure changes, which promotes the expression of IFN-α and TNF-α. HeLa cells secrete IFN-α and TNF-α, which bind to their own receptors and activate the relevant pathways. The cells therefore impact and induce their own cell death, which is an example of autocrine-induced apoptosis. In addition, in vivo experimental data showed that 16


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the reduction of the nano-drug treated tumors was obvious, IFN-α and TNF-α expression significantly increased, and therefore, both in vitro and in vivo studies showed that co-immobilized TNF-α plus IFN-γ induced cancer cell autocrine apoptosis. The discovery of this type of death provides new avenues for the treatment of cervical cancer.

ASSOCIATED CONTENT Supporting Information Supplementary data related to this article can be found at Supplementary-to-manu. The characterization method of co-immobilized TNF-α plus IFN-γ on PSt or Fe3O4-Oleic Acid, including XRD, FRET, contact angle, scanning electron microscope, Confocal Laser Scanning, particle size, IR. The vitro experiments, including western blot. The vivo experiments, construction of tumor nude mice, immunohistochemistry, serological, prussian blue staining.

ACKNOWLEDGMENTS The expenses of this work were supported by the National Natural Science Foundation of China (31370967, 31170919), the Guangdong Province Universities and Colleges Pearl River Scholar Fund Scheme (2014), China, the Science and Technology Planning Project of Guangdong Province (No.2015A020212033), China.

17



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Page 24 of 38

Figure Captions Figure 1. The protein structure diagram of co-immobilized TNF-α plus IFN-γ. Schematic illustration of TNF-α and IFN-γ react with N-(4-AzPhidobenzoyloxy) succinimide to be AzPhTNF-α and AzPhIFN-γ respectively; And the structure interacts in TNF-α and IFN-γ. Schematic illustration of AzPhTNF-α and AzPhIFN-γ through uv irradiation grafted to the surface of polymer material to form PSt-co-immobilized TNF-α plus IFN-γ and AzPhTNF-α and AzPhIFN-γ through uv irradiation grafted to the Fe3O4 to form co-immobilized TNF-α plus IFN-γ-NP.

Figure 2. FRET and X-Ray Powder Diffraction (XRD) testing the structure changes of TNF-α and IFN-γ before and after grafting. (a) Contact angle measuring instrument analysis the crystal structure of PSt-Co-immobilized-TNF-α, PSt-Co-immobilized-IFN-γ, PSt + AzPhTNF-α plus AzPhIFN-γ and PSt-Co-immobilized- TNF-α plus IFN-γ (b) X-Ray Powder Diffraction

(XRD)

analysis

the

crystal

structure

of

PSt-Co-immobilized-TNF-α,

PSt-Co-immobilized-IFN-γ, PSt + AzPhTNF-α plus AzPhIFN-γ and PSt-Co-immobilizedTNF-α plus IFN-γ. (c) Emission spectrum properties of FITC and Rhodamine alone when excited at 495 nm and 565 nm, respectively; and emission of FITC and control Rhodamine when excited at 495 nm (bottom panel). Note that the emission of the acceptor Rhodamine at 565 nm by FITC was increased, whereas no enhanced emission was detected with FITC-Rhodamine. (c1) The image of laser confocal fluorescence. (c2) Fluorescence intensity quantitative column contrast chart. (c3) Fluorescence intensity quantitative analysis diagram.

Figure 3. Characterization of Fe3O4-OA-co-immobilized IFN-γ plus TNF-α. (a) Infrared spectroscopy

analysis

chemical

bond

changes

of

Fe3O4-OA-Immobilized-TNF-α,

Fe3O4-OA-Immobilized-IFN-γ, Fe3O4-OA + AzPhIFN-γ and AzPhTNF-α, co-immobilized TNF-α plus IFN-γ-NP. (b) Particle size detection of Fe3O4-OA-Immobilized-TNF-α, Fe3O4-OA-Immobilized-IFN-γ, Fe3O4-OA-IFN-γ and TNF-α, co-immobilized TNF-α plus IFN-γ-NP, the size of the four nano-drugs were 300 nm, 140 nm, 110 nm, 530 nm. (c) Scanning electron micrograph of Fe3O4-OA-Immobilized-TNF-α, Fe3O4-OA-Immobilized24


Page 25 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


IFN-γ, Fe3O4-OA-IFN-γ and TNF-α, co-immobilized TNF-α plus IFN-γ-NP (The length bar = 500 nm).

Figure 4. Microarray analysis of HeLa cells after induced by co-immobilized TNF-α plus IFN-γ. (a) Microarray of suspected gene related to TNF-α in gene chip in HeLa treated with PSt-co-immobilized TNF-α plus IFN-γ compared with free TNF-α; (b) Respectively, the relative variation of their statistics in HeLa treated with PSt-co-immobilized TNF-α plus IFN-γ compared with PSt + AzPhTNF-α plus AzPhIFN-γ; (c) The changes of TNF-α related genes.

Figure 5. Microarray analysis of HeLa cells after induced by PSt-co-immobilized TNF-α plus IFN-γ. (a) Microarray of suspected gene related to IFN-γ in gene chip in HeLa treated with PSt-co-immobilized TNF-α plus IFN-γ compared with PSt + AzPhTNF-α plus AzPhIFN-γ; (b) Respectively, the relative variation of their statistics in HeLa treated with PSt-co-immobilized TNF-α plus IFN-γ compared with PSt + AzPhTNF-α plus AzPhIFN-γ; (c) The changes of TNF-α related genes. (d) The scatter diagrams (HeLa treated with PSt-co-immobilized TNF-α plus IFN-γ compared with PSt + AzPhTNF-α plus AzPhIFN-γ).

Figure 6. The expression of TNF-α and IFN-α induced by PSt-co-immobilized TNF-α plus IFN-γ. HeLa cells without treatment (CK-group), treated with PSt + AzPhTNF-α plus AzPhIFN-γ (F-group) and PSt-co-immobilized TNF-α plus IFN-γ (C-group). (a)The Illustration of AzPhTNF-α and AzPhIFN-γ were co-immobilized on PSt and acting on Hela cells (b1,b2) Measured mRNA expression data of TNF-α and IFN-α by real-time fluorescent quantitative PCR analysis for HeLa cells. (c) Shows the protein subcellular redistribution data of p TNF-α and IFN-α (white bars: 10 µm) by confocal laser scanning microscope for HeLa cells treated with PSt-co-immobilized TNF-α plus IFN-γ for for different time. (d) TNF-α Elisa Kit and IFN-α Elisa Kit to detected the secretion of TNF-α and IFN-α.

Figure

7.

The

expression

of

TNF-α

and

IFN-α

both

increased

induced

by

PSt-co-immobilized TNF-α plus IFN-γ. And the status of HeLa cell after deal with the 25



inhibitors. (a1) Measured protein expression data of TNF-α and IFN-α by western blotting for HeLa cells without treatment (CK-group), treated with PSt + AzPhTNF-α plus AzPhIFN-γ (F-group) and PSt-co-immobilized IFN-γ plus TNF-α (C-group) TNF-α plus IFN-γ for 12 and 24 h, respectively. (a2,a3)The protein expression was determined using BandScan software. The relative levels are plotted with the significance p < 0.05 labeled by symbol * and p < 0.001 labeled by symbol **, in comparison with the control group. The bars stand for the standard deviations (n = 3). (b1,b2) The expression of IFN-α and TNF-α after the inhibitors(0.2 µmol / L) on HeLa cell. (c1) Cell counting for a rough estimate by flow cytometry, which divided into six categories, respectively is: the blank group, PSt + AzPhTNF-α plus AzPhIFN-γ, PSt-co-immobilized IFN-γ plus TNF-α and blank + inhibitor, PSt + AzPhTNF-α plus AzPhIFN-γ + inhibitor and PSt-co-immobilized IFN-γ plus TNF-α + inhibitor, induce 24 h. (c2) Column chart on flow cytometry results. The relative levels are plotted with the significance p < 0.05 labeled by symbol * and p < 0.001 labeled by symbol **, in comparison with the control group. The bars stand for the standard deviations (n = 3) (d) Changes of cell number in HeLa cells induced by biomaterials. The relative levels are plotted with the significance p < 0.05 labeled by symbol * and p < 0.001 labeled by symbol **, in comparison with the control group. The bars stand for the standard deviations (n = 3).

Figure 8. Effect of nano-drug on tumor progression in nude mice models. (a) Images of tumor-bearing mice treated with the three types of substances: (CK-group) inject normal saline (NS), (F-group) inject Fe3O4-OA + AzPhIFN-γ and AzPhTNF-α, (C-group) inject co-immobilized TNF-α plus IFN-γ-NP. (b) Comparison of tumor changes before and after treatment in three groups of nude mice. (The length bar = 5 cm) (c) The proteins subcellular redistribution data of TNF-α and IFN-α by immunofluorescence for tumor tissue. The length bar = 50 µm. (d) Tumor volume (left), survival rate (middle) and weight (right) of the nude mice as a function of time (days) upon the treatment. (e) Western blotting data of TNF-α and IFN-α in the tumor tissue after treatment.

Figure 9. Effect of nano-drug on tumor progression and toxicities data in nude mice models. (CK-group) inject normal saline (NS), (F-group) inject Fe3O4-OA + AzPhIFN-γ and 26


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AzPhTNF-α, (C-group) inject co-immobilized TNF-α plus IFN-γ-NP (a) Prussian blue staining of the tumor tissues upon the treatment. The length bar = 50 µm. (b) Serological and toxicology test of nude mice, including the density of red blood cell (RBC, left), the density of white blood cell (WBC, middle), and the density of platelet (PLT, right), upon the treatment. Significant increase or decrease at labels (*) (p < 0.05), labels (**) (0.001 < p < 0.01) and labels (***) p < 0.001, are identified in comparison with the CK-group. The bars stand for the standard deviations (n = 6). (c) Prussian blue staining of five normal tissues upon the treatment. The length bar = 100 µm.

Figure 10. Proposed model of regulation by PSt-co-immobilized TNF-α plus IFN-γ biomaterial in cervix cancer cells. (a) The autocrine phenomenon in the synergistic induction by PSt-co-immobilized TNF-α plus IFN-γ involved in our previous findings. (b) PSt-co-immobilized TNF-α plus IFN-γ and co-immobilized TNF-α plus IFN-γ-NP causes autocrine for Hela cell, which causes the occurrence of apoptosis or preventing the division of tumor cells leading to long block. (c) IFN-α produced by Autocrine will bind to the receptor. (d) TNF-α produced by Autocrine will bind to the receptor.

27



Figure 1. The protein structure diagram of co-immobilized TNF-α plus IFN-γ. Schematic illustration of TNF-α and IFN-γ react with N-(4-AzPhidobenzoyloxy) succinimide to be AzPhTNF-α and AzPhIFN-γ respectively; And the structure interacts in TNF-α and IFN-γ. Schematic illustration of AzPhTNF-α and AzPhIFN-γ through uv irradiation grafted to the surface of polymer material to form PSt-co-immobilized TNF-α plus IFN-γ and AzPhTNF-α and AzPhIFN-γ through uv irradiation grafted to the Fe3O4 to form co-immobilized TNF-α plus IFN-γ-NP. 152x180mm (300 x 300 DPI)


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Figure 2. FRET and X-Ray Powder Diffraction (XRD) testing the structure changes of TNF-α and IFN-γ before and after grafting. (a) Contact angle measuring instrument analysis the crystal structure of PSt-Coimmobilized-TNF-α, PSt-Co-immobilized-IFN-γ, PSt + AzPhTNF-α plus AzPhIFN-γ and PSt-Co-immobilizedTNF-α plus IFN-γ (b) X-Ray Powder Diffraction (XRD) analysis the crystal structure of PSt-Co-immobilizedTNF-α, PSt-Co-immobilized-IFN-γ, PSt + AzPhTNF-α plus AzPhIFN-γ and PSt-Co-immobilized- TNF-α plus IFN-γ. (c) Emission spectrum properties of FITC and Rhodamine alone when excited at 495 nm and 565 nm, respectively. (c1) The image of laser confocal fluorescence. (c2) Fluorescence intensity quantitative column contrast chart. (c3) Fluorescence intensity quantitative analysis diagram. 152x178mm (300 x 300 DPI)



Figure 3. Characterization of Fe3O4-OA-co-immobilized IFN-γ plus TNF-α. (a) Infrared spectroscopy analysis chemical bond changes of Fe3O4-OA-Immobilized-TNF-α, Fe3O4-OA-Immobilized-IFN-γ, Fe3O4-OA + AzPhIFN-γ and AzPhTNF-α, co-immobilized TNF-α plus IFN-γ-NP. (b) Particle size detection of Fe3O4-OAImmobilized-TNF-α, Fe3O4-OA-Immobilized-IFN-γ, Fe3O4-OA-IFN-γ and TNF-α, co-immobilized TNF-α plus IFN-γ-NP, the size of the four nano-drugs were 300 nm, 140 nm, 110 nm, 530 nm. (c) Scanning electron micrograph of Fe3O4-OA-Immobilized-TNF-α, Fe3O4-OA-Immobilized- IFN-γ, Fe3O4-OA-IFN-γ and TNF-α, co-immobilized TNF-α plus IFN-γ-NP (The length bar = 500 nm). 211x188mm (300 x 300 DPI)


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Figure 4. Microarray analysis of HeLa cells after induced by co-immobilized TNF-α plus IFN-γ. (a) Microarray of suspected gene related to TNF-α in gene chip in HeLa treated with PSt-co-immobilized TNF-α plus IFN-γ compared with free TNF-α; (b) Respectively, the relative variation of their statistics in HeLa treated with PSt-co-immobilized TNF-α plus IFN-γ compared with PSt + AzPhTNF-α plus AzPhIFN-γ; (c) The changes of TNF-α related genes. 119x131mm (300 x 300 DPI)



Figure 5. Microarray analysis of HeLa cells after induced by PSt-co-immobilized TNF-α plus IFN-γ. (a) Microarray of suspected gene related to IFN-γ in gene chip in HeLa treated with PSt-co-immobilized TNF-α plus IFN-γ compared with PSt + AzPhTNF-α plus AzPhIFN-γ; (b) Respectively, the relative variation of their statistics in HeLa treated with PSt-co-immobilized TNF-α plus IFN-γ compared with PSt + AzPhTNF-α plus AzPhIFN-γ; (c) The changes of TNF-α related genes. (d) The scatter diagrams (HeLa treated with PSt-coimmobilized TNF-α plus IFN-γ compared with PSt + AzPhTNF-α plus AzPhIFN-γ). 119x142mm (300 x 300 DPI)


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Figure 6. The expression of TNF-α and IFN-α induced by PSt-co-immobilized TNF-α plus IFN-γ. HeLa cells without treatment (CK-group), treated with PSt + AzPhTNF-α plus AzPhIFN-γ (F-group) and PSt-coimmobilized TNF-α plus IFN-γ (C-group). (a)The Illustration of AzPhTNF-α and AzPhIFN-γ were coimmobilized on PSt and acting on Hela cells (b1,b2) Measured mRNA expression data of TNF-α and IFN-α by real-time fluorescent quantitative PCR analysis for HeLa cells. (c) Shows the protein subcellular redistribution data of p TNF-α and IFN-α (white bars: 10 µm) by confocal laser scanning microscope for HeLa cells treated with PSt-co-immobilized TNF-α plus IFN-γ for for different time. (d) TNF-α Elisa Kit and IFN-α Elisa Kit to detected the secretion of TNF-α and IFN-α. 152x218mm (300 x 300 DPI)



Figure 7. The expression of TNF-α and IFN-α both increased induced by PSt-co-immobilized TNF-α plus IFN γ. And the status of HeLa cell after deal with the inhibitors. (a1) Measured protein expression data of TNF- α and IFN-α by western blotting for HeLa cells without treatment (CK-group), treated with PSt + AzPhTNF-α plus AzPhIFN-γ (F-group) and PSt-co-immobilized IFN-γ plus TNF-α (C-group) TNF-α plus IFN-γ for 12 and 24 h, respectively. (a2,a3)The protein expression was determined using BandScan software. (b1,b2) The expression of IFN-α and TNF-α after the inhibitors(0.2 µmol / L) on HeLa cell. (c1) Cell counting for a rough estimate by flow cytometry, which divided into six categories, induce 24 h. (c2) Column chart on flow cytometry results. (d) Changes of cell number in HeLa cells induced by biomaterials. 211x273mm (300 x 300 DPI)


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Figure 8. Effect of nano-drug on tumor progression in nude mice models. (a) Images of tumor-bearing mice treated with the three types of substances: (CK-group) inject normal saline (NS), (F-group) inject Fe3O4-OA + AzPhIFN-γ and AzPhTNF-α, (C-group) inject co-immobilized TNF-α plus IFN-γ-NP. (b) Comparison of tumor changes before and after treatment in three groups of nude mice. (The length bar = 5 cm) (c) The proteins subcellular redistribution data of TNF-α and IFN-α by immunofluorescence for tumor tissue. The length bar = 50 µm. (d) Tumor volume (left), survival rate (middle) and weight (right) of the nude mice as a function of time (days) upon the treatment. (e) Western blotting data of TNF-α and IFN-α in the tumor tissue after treatment. 152x202mm (300 x 300 DPI)



Figure 9. Effect of nano-drug on tumor progression and toxicities data in nude mice models. (CK-group) inject normal saline (NS), (F-group) inject Fe3O4-OA + AzPhIFN-γ and AzPhTNF-α, (C-group) inject coimmobilized TNF-α plus IFN-γ-NP (a) Prussian blue staining of the tumor tissues upon the treatment. The length bar = 50 µm. (b) Serological and toxicology test of nude mice, including the density of red blood cell (RBC, left), the density of white blood cell (WBC, middle), and the density of platelet (PLT, right), upon the treatment. Significant increase or decrease at labels (*) (p < 0.05), labels (**) (0.001 < p < 0.01) and labels (***) p < 0.001, are identified in comparison with the CK-group. The bars stand for the standard deviations (n = 6). (c) Prussian blue staining of five normal tissues upon the treatment. The length bar = 100 µm. 152x189mm (300 x 300 DPI)


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Figure 10. Proposed model of regulation by PSt-co-immobilized TNF-α plus IFN-γ biomaterial in cervix cancer cells. (a) The autocrine phenomenon in the synergistic induction by PSt-co-immobilized TNF-α plus IFN-γ involved in our previous findings. (b) PSt-co-immobilized TNF-α plus IFN-γ and co-immobilized TNF-α plus IFN-γ-NP causes autocrine for Hela cell, which causes the occurrence of apoptosis or preventing the division of tumor cells leading to long block. (c) IFN-α produced by Autocrine will bind to the receptor. (d) TNF-α produced by Autocrine will bind to the receptor. 169x153mm (300 x 300 DPI)



TOC graphic 198x85mm (300 x 300 DPI)


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Cervical Cancer HeLa Cell Autocrine Apoptosis Induced by Co

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