Prodrug-Like, PEGylated Protein Toxin Trichosanthin for Reversal of

Feb 14, 2017 - Multidrug resistance (MDR) is a main obstacle in cancer chemotherapy. The MDR mechanisms involve P-glycoprotein (P-gp) overexpression, ...
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Prodrug-Like, PEGylated Protein Toxin Trichosanthin for Reversal of Chemoresistance Yingzhi Chen,†,‡ Meng Zhang,†,‡ Hongyue Jin,†,‡ Yisi Tang,§ Aihua Wu,§ Qin Xu,§ and Yongzhuo Huang*,‡ †

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Hai-ke Rd, Shanghai 201203, China University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China § Guangzhou University of Chinese Medicine, Tropical Medical Institute, 12 Ji-chang Rd, Guangzhou 510450, China ‡

S Supporting Information *

ABSTRACT: Multidrug resistance (MDR) is a main obstacle in cancer chemotherapy. The MDR mechanisms involve P-glycoprotein (P-gp) overexpression, abnormality of apoptosis-related protein, and altered expression of drug-targeting proteins. Therapeutic proteins are emerging as candidates for overcoming cancer MDR because of not only their large molecular size that potentially circumvents the P-gp-mediated drug efflux but also their distinctive bioactivity distinguished from small-molecular drugs. Herein we report trichosanthin, a plant protein toxin, possesses synergistic effect with paclitaxel (PTX) in the PTX-resistance A549/T nonsmall cell lung cancer (NSCLC) cells, by reversing PTX-caused caspase 9 phosphorylation and inducing caspase 3-dependent apoptosis. Moreover, via intein-mediated site-specific protein ligation, a matrix metalloproteinase (MMP)-activatable cell-penetrating trichosanthin delivery system was constructed by modification of a cell-penetrating peptide and MMP-2-sensitive PEGylation to overcome the limitation of in vivo application of trichosanthin, by improving the short half-life and poor tumor targeting, as well as immunogenicity. In a mouse model bearing A549/T tumor, the MMP-activatable trichosanthin was further tested for its application for MDR reversal in combination with PTX liposomes. The delivery system showed synergy effect with PTX-loaded liposome in treating MDR cancer in vivo. KEYWORDS: multidrug resistance, trichosanthin, matrix metalloproteinase, paclitaxel, caspase 9 phosphorylation, intein-mediated protein ligation, PEGylation

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INTRODUCTION

evaluation has been an important direction in precision medicine. Ribosome-inactivating proteins (RIPs), derived from plants or bacteria, are a class of emerging antitumor agents that possess N-glycosidase activity to inhibit protein synthesis.10 Type I is a class of most investigated RIPs, which have been tested in clinical trials and displayed encouraging potential.10 RIPs have very high in vitro antitumor activity,11 and one RIP molecule can kill one tumor cell as long as it enters cytosol.12 RIPs (e.g., trichosanthin, TCS) can induce cell apoptosis through various mechanisms.13−16 To our interest, RIPs have been reported with ability to kill MDR cancer cells.17 However, there is little information available on the anti-MDR mechanisms of RIPs, and there are few reports of RIPs on anti-MDR treatment in vivo yet. We thus launched an investigation on this issue, in an effort to develop a potential

Multidrug resistance (MDR) is the major challenge against effective cancer treatment by causing failure of chemotherapy. The mechanisms involved in MDR are complicated and generally associated with overexpression of P-glycoprotein (Pgp),1 abnormality of apoptosis pathways,2 the altered expression of drug-targeting proteins,3 and so on. It is a pressing need to seek for potential solutions for overcoming MDR. For example, development of innovative drugs with new targets has been actively pursued. However, the processes require science and regulation to advance in concert,4 letting along the skyrocketing cost and decades-long efforts. Nanotechnology-based strategies for combating P-gp-overexpressioncaused tumor MDR have been widely explored.5,6 For instance, application of drug-loaded nanoparticles modified with cellpenetrating peptides (CPP) to bypass drug efflux and increase intratumoral penetration, cellular uptake, and nuclear delivery are the effective approaches to overcome MDR.7,8 In addition, it is also well established that combination therapy is a useful strategy to reverse MDR.9 A search for new drug combination in treating tumor MDR via different mechanisms for preclinical © XXXX American Chemical Society

Special Issue: Bioconjugate Therapeutics Received: November 1, 2016 Revised: January 26, 2017 Accepted: January 30, 2017

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DOI: 10.1021/acs.molpharmaceut.6b00987 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 1. Protein expression, purification, and modification. (A) Scheme of protein C-terminal PEGylation using IPL strategy. (B) Scheme of recombinant plasmid construction, rTCS and rTLM. (C) Synthesis of the rTLM-PEG conjugate using IPL strategy. (D) SDS-PAGE electrophoresis of the rTCS and rTLM-PEG. Lane M, standard protein marker. Lane 1, rTCS. Lane 2, rTLM-PEG. (E) Circular dichroism and (F) size exclusion chromatography analysis of the rTCS and rTLM-PEG conjugate.

investigated both in vitro and in vivo for its synergistic effect on anti-MDR cancer in combination with paclitaxel.

mediation for overcoming MDR cancer, with a focus on combination effect of RIPs and small-molecular chemo drugs. Despite their potent activity, the clinical translation of RIPs is impeded by the poor drug-like property (e.g., short half-life, insufficient tumor accumulation and cell penetration, and strong immunogenicity).18 We thereby developed a tumortargeting, prodrug-like RIP delivery system with improved druggability in which the recombinant trichosanthin (TCS) with fusion with a cell-penetrating peptide (CPP) and matrix metalloproteinase (MMP) substrate peptide was site-specifically PEGylated at C-terminus based on intein-mediated protein ligation (IPL) technique. IPL technique was developed for untagged protein expression and labeling. It, however, has rarely been reported for construction of protein drug delivery system. By combining genetic engineering and organic synthesis, the tumor-targeted MMP-2-activatable rTCS delivery system was constructed by intein-mediated site-specific PEGylation. The PEGylated CPP-TCS displayed a prodruglike feature due to its inability to cell penetration, unless PEG was cleaved by the tumor-associated gelatinases. The system showed extended half-life, improved tumor accumulation, and enhanced antitumor efficacy in MMP-2-overexpressing tumor model, which was described in our previous work.19 Herein, the enhanced delivery system of PEGylated CPP-TCS was

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RESULTS Protein Preparation and Characterization. Site-specific modification of proteins has been a challenge for protein modification and protein drug industry. We here used the intein-mediated protein ligation strategy for C-terminal sitespecific PEGylation. Intein-based technique is characterized by its facile process in which the recombinant proteins can be purified through chitin affinity chromatography and then activated by intein-mediated, cysteine-induced on-column cleavage, thus yielding a C-terminal cysteine for further thiolbased conjugation (Figure 1A). TCS is a type I RIP with MW about 27 kDa, which is derived from the Chinese herb Tian Hua Fen (the root of Trichosanthes kirilowii Maxim).10 The recombinant plasmid pTLM was constructed by using the vector pTXB1 to produce the fusion protein that contains a TCS with C-terminal CPP sequence (low-molecular weight protamine, LMWP) and MMP-2 substrate peptide (MSP), termed rTLM (Figure 1B). The plasmid pTCS encoding rTCS (without CPP sequence) was prepared as a control for further studies. B

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Figure 2. MMP-2-mediated cleavage of the rTLM-PEG. (A) Western blot detection of cellular MMP-2 level of A549/T cells compared with MMP2-positive control HT1080 cells. (B) Gelatin zymography assay of HT1080 and A549/T-conditioned medium. (C) Cleavage of the rTLM-PEG by A549/T-conditioned medium.

Figure 3. MMP-2-mediated cellular uptake in A549/T cells. (A) Fluorescent imaging and (B,C) flow cytometry analysis of A549/T cells incubated with FITC-labeled uncleaved rTLM-PEG, cleaved rTLM-PEG, and cleaved rTLM-PEG preincubated with heparin (***p < 0.001).

Both proteins were purified by chitin affinity chromatography and then cleaved from the column by using eluting buffer

containing cysteine. The product rTLM was then conjugated with maleimide-PEG5k (Mal-PEG) to form the rTLM-PEG C

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Figure 4. In vitro cytotoxicity. (A) Cytotoxicity of the rTCS and rTLM-PEG conjugate on A549/T cells alone or with the addition of 20 μg/mL PTX. (B) Cytotoxicity of PTX in A549/T cells alone or with the addition of 1 μM rTCS or rTLM-PEG. (C) The overall combination effect of the two protein drugs and PTX in A549/T cells (***p < 0.001).

FITC-labeled rTLM-PEG was precleaved by A549/Tconditioned medium to test its MMP-2-mediated cellular uptake on A549/T cells compared with its uncleaved control. As shown in Figure 3, the precleaved rTLM-PEG had higher cellular uptake level than that without precleavage treatment, demonstrated by both fluorescent imaging and FACS analysis, indicating MMP-2-sensitive, selective delivery of rTLM-PEG. In addition, the cell penetration of the cleaved rTLM-PEG was inhibited by heparin because the exposed cationic CPP was neutralized by the polyanionic heparin through charge interaction. This further demonstrated the enhanced cellular uptake of the cleaved rTLM-PEG was a result of the exposure of CPP. In Vitro Cytotoxicity in A549/T Cells. The in vitro cytotoxicity of the prodrug-like delivery system rTLM-PEG was tested on PTX-resistant human NSCLC A549/T cells in combination with PTX. It was shown that rTLM-PEG had enhanced cytotoxicity compared to the unmodified rTCS (Figure 4A), due to the MMP-2-mediated dePEGylation to release CPP-fused rTCS during the 48-h incubation. However, both the rTCS and rTLM-PEG showed potent anti-MDR cytotoxicity in combination of 20 μg/mL PTX (IC50 58 versus 23 nM), yielding synergistic effect compared to single use of proteins (IC50, 2430 versus 1530 nM) (Figure 4A and Table 1). Meanwhile, the cell viability remained over 60% when the cells were treated with 20 μg/mL PTX. Although A549/T cells were resistant to PTX, our results demonstrated the combination therapy could resensitize the cells to PTX. With addition of 1 μM rTCS or rTLM-PEG, the IC50 value of PTX in A549/T cells dropped by 5.4- and 16.2fold, respectively (Figure 4B and Table 2). The overall combination effect of the protein drugs and PTX in A549/T

conjugate (Figure 1C). The rTLM-PEG conjugate was characterized and analyzed by SDS-PAGE electrophoresis, showing the purity of rTCS was over 90% and the PEGylation efficiency of rTLM-PEG was over 85% (Figure 1D). The rTLM-PEG conjugate was then characterized using matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), circular dichroism (CD), and size exclusion chromatography (SEC). MALDI-TOF-MS results showed molecular weight of the rTCS and rTLMPEG were 27.8 and 36.2 kDa, respectively, consistent with their theoretical molecular weight (Figure S1). CD analysis showed the similar secondary structure of rTLM-PEG and rTCS (Figure 1E). SEC showed over 90% purity of both proteins. In addition, the rTLM-PEG exhibited shorter retention time than rTCS, indicating the increased hydraulic size of rTLM-PEG (Figure 1F). MMP-2-Mediated Cleavage of rTLM-PEG and Cellular Uptake. The rTLM-PEG was designed to be specifically cleaved and activated by tumor-associated matrix metalloproteinases (MMPs), as we previously reported that rTLMPEG showed less cytotoxicity on MMP-2-negative HUVEC cells compared that on MMP-2-positive tumor cells in our previous work; in addition, an MMP-2 noncleaved conjugate rTL-PEG showed much less toxicity than rTLM-PEG in MMP2-positive tumor cells.19 In the PTX-resistant A549/T cells, high level of MMP-2 was detected in both cytosol and cell medium (Figure 2A). In addition, high MMP activity was also detected in A549/T-conditioned cell medium using gelatin zymography assay (Figure 2B). The rTLM-PEG exposed to the A549/T-conditioned medium was efficiently cleaved (Figure 2C), demonstrating the feasibility of MMP-2-mediated activation of the rTLM-PEG. D

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arrested the drug-resistant tumor growth (Figure 6A,B). By contrast, single use of the rTLM-PEG or PTX liposomes at the same dose did not yield sufficient treatment outcomes. Moreover, the combination therapy did not cause significant change in body weight, indicating the good tolerance and reduced side toxicity (Figure 6C). In the xenografted A549/T tumor, there was overexpression of both MMP-9 and MMP-2 while only high level of MMP-2 was found in the HT1080 tumor (Figure 6D). The MSP linker in rTLM-PEG was susceptible to both MMP-9 and MMP-2. Therefore, this prodrug-like system was favorable for targeting the drugresistant A549/T tumor. Of note, the MMP-2 level in tumor tissues in higher than that in cell medium (Figure 6D). Expression of the tumorassociated proteases are regulated by tumor microenvironment, and the levels in tumor tissues are usually much higher than that in cultured cells.21 It allowed the rTLM-PEG to be much efficiently cleaved and activated in the in vivo treatment without pretreatment. In addition, the tumor tissues were collected for immunohistochemical (IHC) staining of cleaved caspase 3 and TUNEL staining to investigate the apoptosis inside tumors. Cleaved caspase 3, representing early apoptosis, was shown to have a much higher level in the combination therapy group than the other monotherapy groups (Figure 7A). The results were consistent with the elevated cellular level of cleaved caspase 3 after the combination treatment in Figure 5. Similarly, the TUNEL level, indicating late apoptosis, was also higher in the combination therapy group, whereas the single use of rTLM-PEG or PTX liposomes showed relatively lower level in the tumors (Figure 7B). Elevated cleaved caspase 3 and TUNEL level demonstrated the potency of anti-MDR cancer treatment by using the combination therapy of rTLM-PEG and PTX liposome.

Table 1. IC50 Values of rTCS and rTLM-PEG Alone or with 20 μg/mL PTX in A549/T Cells IC50 (nM) rTCS rTLM-PEG

alone

with PTX (20 μg/mL)

fold

2430 1575

58.22 23.17

42 68

Table 2. IC50 Values of PTX Alone or with 1 μM rTCS or rTLM-PEG in A549/T Cells IC50 (μg/mL) PTX fold

alone

with rTCS (1 μM)

with rTLM-PEG (1 μM)

25.9

4.8 5.4

1.6 16.2

cells was shown in Figure 4C. The combination index (CI) of rTCS with PTX was 0.6, and rTLM-PEG with PTX was 0.7. Mechanism for rTCS for MDR Reversal. So far, there is little information available on the TCS-mediated reversal of MDR. Therefore, we applied protein microarray analysis for monitoring the apoptosis-related protein expression. The results showed phosphorylation of caspase 9 was significantly up-regulated (Figure S2, Table 3). Caspase 9 plays a crucial role Table 3. Caspase 9 Phosphorylation Level Change Detected by Protein Microarray Screening name caspase 9 (phosphoSer144) caspase 9 (phosphoSer196) caspase 9 (phosphoThr125) caspase 9 (phosphoTyr153)

blank phospho/ unphos

PTX phospho/ unphos

PTX + rTCS phospho/unphos

1.08

0.88

0.33

0.43

0.40

0.32

1.58

5.50

1.44

1.33

1.50

1.37

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DISCUSSION Different strategies for combating MDR cancer have been widely investigated. The involvement of altered apoptosisrelated protein level is also a crucial factor to cause cancer cells apoptosis resistance to chemotherapy.2 Combination therapy is the major strategy to overcome MDR cancer. Search for effective drug combination and understanding their synergistic mechanisms are pressing needs for developing successful therapies against MDR cancer. Application of RIPs in combating MDR has rarely been reported. We found TCS could reverse drug-resistance in A549/T cells and resensitize them to PTX. The antibody microarray and Western blotting results revealed the MDR reversal was associated with suppression of caspase 9 phosphorylation. Phosphorylation of caspase 9 has been reported as a negative indicator to the apoptotic fate of cells treated with antimitosis drugs such as taxol, and inhibition of caspase 9 phosphorylation can cause resensitization of cancer cells to chemo drugs.22 For example, enhanced activity of caspase 9 is related to inhibition of phosphorylation at Ser144.23 Therefore, caspase 9 is considered as an important therapeutic target for cancer therapy as well as reversal of drug resistance.24 However, the mechanisms of MDR are complicated, and there is still much unknown about TCS in combating PTX resistance. Our results revealed one of the many possible mechanisms, but further investigation should be conducted to acquire more detailed information. However, it

in cell apoptosis; its activation can be inhibited through phosphorylation at multiple sites.20 Based on that, caspase 9 phosphorylation level on various sites was determined by protein microarray. The results showed that caspase 9 phosphorylation on Thr125 was elevated in A549/T cells treated with PTX alone, but the phosphorylation on Ser144 and Thr125 was suppressed by coapplication of TCS and PTX, which was further confirmed by Western blotting (Figure 5A). As a result, caspase 9 activity increased after combination treatment with PTX and TCS (Figure 5C), which further led to the downstream activation of caspase 3 (Figure 5B,D). It was indicated thereby that PTX-induced caspase 9 phosphorylation on Thr125 was a potential mechanism for PTX resistance in A549/T cells and demonstrated TCS could reverse drug resistance through suppression of caspase 9 phosphorylation, which consequently activated the downstream caspase 3 and induced apoptosis. In Vivo Anti-MDR Cancer Treatment. The investigation of anti-MDR treatment efficacy was carried out in a drugresistant A549/T tumor animal model by giving the rTLMPEG and PTX liposomes. The rTLM-PEG successfully reversed MDR, and the combination therapy completely E

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Figure 5. Mechanism study of TCS for reversal of PTX-resistance. (A) Caspase 9 and its phosphorylation level, and (B) caspase 3 and cleaved caspase 3 level detection. (C) Caspase 9 and (D) caspase 3 activity measurement.

Figure 6. In vivo anti-MDR cancer treatment. (A) Tumor growth curve during the treatment. (B) Tumor images at the end point of the treatment. (C) Animal body weight change during the treatment. (D) MMP-2 and MMP-9 level detection in A549/T tumor and HT1080 tumor (***p < 0.001).

provided convincing support for the potential of combination therapy of TCS and PTX in application of anti-MDR cancer.

Although TCS has potent antitumor activity, there are several bottlenecks that constrain its application. First, TCS F

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efficiency for enhancing treatment yet reducing side toxicity. For example, use of a targeting ligand to modify PEG would further facilitate the targeting, or the specificity of CPPmediated delivery could be improved by use of charge-reversal strategy by responding to tumor microenvironments (e.g., ROS and enzymes29,30). Lastly, it should be mentioned that, although proteaseactivatable strategy has been actively explored in nanotechnology-based drug delivery, there are very few investigation in protein delivery. Our results revealed that this strategy provides a promising solution to the cytoplasmic protein drugs.

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CONCLUSIONS In summary, we used intein-based site-specific modification to construct a prodrug-like gelatinase-responsive rTCS toxin delivery system, which could be cleaved and activated by tumor-specific MMPs to achieve protease-mediated cellular uptake and cytotoxicity. The system showed combination effect with PTX in PTX-resistant NSCLC treatment both in vitro and in vivo. In addition, the mechanism for rTCS toxin in reversal of PTX resistance was investigated using antibody microarray, showing rTCS inhibited PTX-caused elevated caspase 9 phosphorylation to activate caspase 3 and induce apoptosis. The combination of a protein toxin and a small molecular chemo drug offers a potential solution to anti-MDR cancer therapy.

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EXPERIMENTAL SECTION Materials. The original TCS-expressing plasmid28 was kindly provided by Prof. Pang-Chui Shaw, The Chinese University of Hong Kong. E. coli strain BL21 (DE3) was preserved by our laboratory. The IMPACT (Intein-mediated purification with affinity chitin-binding tag) system, including the expressing vector pTXB1 and chitin resin, was acquired from New England Biolabs (U.K.). Lysogeny Broth (LB) medium was purchased from Oxoid (U.K.). Maleimide-PEG5k was purchased from Jenkem Technology Co., Ltd. (Beijing, China). Standard protein markers and isopropyl β-D-1thiogalactopyranoside (IPTG) were acquired from Thermo Scientific (USA). BCA microplate protein assay kit was obtained from Beyotime Institute of Biotechnology (Haimen, China). 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and cocktail protease inhibitor were purchased from Sigma-Aldrich Co., Ltd. (USA). Fetal bovine serums (FBS), RPMI 1640 medium, Dulbecco’s modified Eagle’s Medium (DMEM) cell culture medium and 0.25% trypsin-EDTA were purchased from Gibco (USA). All used antibiotics and bovine serum albumin (BSA) were acquired from Amresco (USA). L-Cysteine was obtained from J&K Scientific Ltd. (Shanghai, China). PTX was obtained from Melonepharma Biotechnology Co., Ltd. (Dalian, China). Soybean phosphatidylcholine (SPC), cholesterol (CHOL), and DSPE-PEG2k were purchased from AVT Medical Science Ltd. (Shanghai, China). All other reagents were of analytical grade from Sinapharm Chemical Reagent Co., Ltd. (Shanghai, China). Protein Expression. The sequence of low molecular weight protamine (LMWP, VSRRRRRRGGRRRR) and MMP-2 substrate peptide (MSP, PLGLAG) were added to the terminal of TCS by polymerase chain reaction (PCR) to prepare the recombinant gene for the fusion protein recombinant TCSLMWP-MSP (rTLM). Both TCS and TLM sequence were

Figure 7. Apoptosis detection inside the tumors. (A) Immunohistochemical staining of cleaved caspase 3 and (B) TUNEL fluorescent staining of the tumor tissue.

lacks cell permeability and in vivo tumor targeting.25 Second, it has short circulation half-life because small-size proteins (typically 3). Statistical analyses were conducted using unpaired Student’s t test (for comparison between two groups) or oneway ANOVA test (for comparison between more than three groups) by GraphPad Prism. *p < 0.05; **p < 0.01; ***p < 0.001.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.6b00987. MALDI-TOF-MS analysis of the rTCS and rTLM-PEG conjugate; protein microarray screening (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel +86-21-20231000, ext. 1401. Fax: +86-21-20231981. Email: [email protected]. ORCID

Yongzhuo Huang: 0000-0001-7067-8915 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by 973 Program, China (2014CB931900, 2013CB932503) and NSFC, China (81172996, 81422048, 81521005, 81673382). The original TCS plasmid was kindly provided by Prof. Pang-Chui Shaw, The Chinese University of Hong Kong. The MALDI-TOF-MS analysis was performed at the National Center for Protein Science Shanghai, CAS. I

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(17) Selbo, P. K.; Weyergang, A.; Bonsted, A.; Bown, S. G.; Berg, K. Photochemical internalization of therapeutic macromolecular agents: a novel strategy to kill multidrug-resistant cancer cells. J. Pharmacol. Exp. Ther. 2006, 319, 604−12. (18) Shin, M. C.; Zhang, J.; Min, K. A.; He, H.; David, A. E.; Huang, Y.; Yang, V. C. PTD-Modified ATTEMPTS for Enhanced Toxinbased Cancer Therapy: An In Vivo Proof-of-Concept Study. Pharm. Res. 2015, 32, 2690−703. (19) Chen, Y.; Zhang, M.; Jin, H.; Tang, Y.; Wang, H.; Xu, Q.; Li, Y.; Li, F.; Huang, Y. Intein-mediated site-specific synthesis of tumortargeting protein delivery system: Turning PEG dilemma into prodrug-like feature. Biomaterials 2017, 116, 57−68. (20) Allan, L. A.; Clarke, P. R. Apoptosis and autophagy: Regulation of caspase-9 by phosphorylation. FEBS J. 2009, 276, 6063−73. (21) Jiang, Y.; Lu, J.; Wang, Y.; Zeng, F.; Wang, H.; Peng, H.; Huang, M.; Jiang, H.; Luo, C.; Huang, Y. Molecular-dynamics-simulationdriven design of a protease-responsive probe for in-vivo tumor imaging. Adv. Mater. 2014, 26, 8174−8. (22) Allan, L. A.; Clarke, P. R. Phosphorylation of caspase-9 by CDK1/cyclin B1 protects mitotic cells against apoptosis. Mol. Cell 2007, 26, 301−10. (23) Brady, S. C.; Allan, L. A.; Clarke, P. R. Regulation of caspase 9 through phosphorylation by protein kinase C zeta in response to hyperosmotic stress. Mol. Cell. Biol. 2005, 25 (23), 10543−55. (24) Kim, B.; Srivastava, S. K.; Kim, S. H. Caspase-9 as a therapeutic target for treating cancer. Expert Opin. Ther. Targets 2015, 19, 113−27. (25) Sha, O.; Niu, J.; Ng, T. B.; Cho, E. Y.; Fu, X.; Jiang, W. Antitumor action of trichosanthin, a type 1 ribosome-inactivating protein, employed in traditional Chinese medicine: a mini review. Cancer Chemother. Pharmacol. 2013, 71, 1387−93. (26) An, Q.; Lei, Y.; Jia, N.; Zhang, X.; Bai, Y.; Yi, J.; Chen, R.; Xia, A.; Yang, J.; Wei, S.; Cheng, X.; Fan, A.; Mu, S.; Xu, Z. Effect of sitedirected PEGylation of trichosanthin on its biological activity, immunogenicity, and pharmacokinetics. Biomol. Eng. 2007, 24, 643−9. (27) Hatakeyama, H.; Akita, H.; Harashima, H. The polyethyleneglycol dilemma: advantage and disadvantage of PEGylation of liposomes for systemic genes and nucleic acids delivery to tumors. Biol. Pharm. Bull. 2013, 36, 892−9. (28) Zhu, R. H.; Ng, T. B.; Yeung, H. W.; Shaw, P. C. High level synthesis of biologically active recombinant trichosanthin in Escherichia coli. Int. J. Pept. Protein Res. 1992, 39, 77−81. (29) Liu, X.; Xiang, J.; Zhu, D.; Jiang, L.; Zhou, Z.; Tang, J.; Liu, X.; Huang, Y.; Shen, Y. Fusogenic Reactive Oxygen Species Triggered Charge-Reversal Vector for Effective Gene Delivery. Adv. Mater. 2016, 28, 1743−52. (30) Qiu, N.; Liu, X.; Zhong, Y.; Zhou, Z.; Piao, Y.; Miao, L.; Zhang, Q.; Tang, J.; Huang, L.; Shen, Y. Esterase-activated charge-reversal polymer for fibroblast-exempt cancer gene therapy. Adv. Mater. 2016, 28, 10613−10622.

ABBREVIATIONS BCA, bicinchoninic acid; CBD, chitin-binding domain; CPP, cell-penetrating peptide; Cys, cysteine; DMSO, dichloromethane; FITC, fluorescein isothiocyanate; FPLC, fast protein liquid chromatography; IPL, intein-mediated protein ligation; IPTG, Isopropyl β-D-thiogalactoside; LMWP, low molecularweight protamine; Mal, maleimide; MALDI-TOF-MS, matrixassisted laser desorption/ionization time-of-flight mass spectrometry; MDR, multidrug resistance; MMP-2, matrix metalloproteinase-2; MMP-9, matrix metalloproteinase-9; MSP, MMP-2 substrate peptide; MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide; SDS-PAGE, sodium dodecyl sulfonate-polyacrylamide gel electrophoresis; PBS, phosphate buffered saline; PEG, polyethylene glycol; PTX, paclitaxel; TCS, trichosanthin

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