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Phthalazino[1,2-b]quinazolinones as p53 Activators: Cell Cycle Arrest, Apoptotic Response and Bak–Bclxl Complex Reorganization in Bladder Cancer Cells Guo-Hai Zhang, Jing-Mei Yuan, Gang Qian, Chen-Xi GU, Kai Wei, Dong-Liang Mo, Jiang-Ke Qin, Yan Peng, Zuping Zhou, Cheng-Xue Pan, and Gui-Fa Su J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01769 • Publication Date (Web): 26 Jul 2017 Downloaded from http://pubs.acs.org on July 26, 2017

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Journal of Medicinal Chemistry

Phthalazino[1,2-b]quinazolinones as p53 Activators: Cell Cycle Arrest, Apoptotic Response and Bak–Bcl-xl Complex Reorganization in Bladder Cancer Cells

Guo-Hai Zhang†,‡,#, Jing-Mei Yuan†,#, Gang Qian†, Chen-Xi Gu†, Kai Wei†, Dong-Liang Mo†, Jiang-Ke Qin†, Yan Peng†, Zu-Ping Zhou‡, Cheng-Xue Pan*,†, Gui-Fa Su*,†

†

State Key Laboratory for Chemistry and Molecular Engineering of Medicinal

Resources, Ministry of Science and Technology of China; School of Chemistry and Pharmaceutical Sciences, Guangxi Normal University, 15 Yu Cai Road, Guilin 541004, China. ‡

Guangxi Universities Key Laboratory of Stem Cell and Biopharmaceutical

Technology, School of Life Sciences, Guangxi Normal University, 15 Yu Cai Road, Guilin 541004, China. TITLE RUNNING HEAD: Quinazolinones as p53 activators KEYWORDS: quinazolinones, p53, conformational activation of Bak, apoptosis

*Corresponding Authors: Professors Cheng-Xue Pan and Gui-Fa Su Tel. & Fax: +86 773 5826869, Email: [email protected] (C.-X. Pan); [email protected] (G.-F. Su) 1

ACS Paragon Plus Environment


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ABSTRACT: p53 inactivation is a clinically defined characteristic for cancer treatment-nonresponsiveness. It is therefore highly desirable to develop anticancer agents by restoring p53 function.1 Herein the synthesized phthalazino[1,2-b]-quinazolinones were discovered as p53 activators in bladder cancer cells. 10-Bromo-5-(2-dimethylamino-ethylamino)phthalazino[1,2-b]quinazolin-8-one (5da) was identified as the most promising candidate in view of both its anticancer activity and mechanisms of action. 5da exhibited strong anticancer activity on a broad range of cancer cell lines and significantly reduced tumor growth in xenograft models at doses as low as 6 mg/kg. Furthermore, 5da caused cell cycle arrest at S/G2 phase, induced apoptosis, changed cell size and led to cell death by increasing the proportion of sub-G1 cells. Molecular mechanism studies suggested that accumulation of phospho-p53 in mitochondria after 5da treatment resulted in conformational activation of Bak, thereby evoking cell apoptosis, finally leading to irreversible cancer cell inhibition. Our present studies furnish new insights into the molecular interactions and anticancer mechanisms of phospho-p53-dependent quinazolinone compound.

2


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INTRODUCTION Bladder cancer (BC) continues to be the second most common malignancy of urinary system worldwide with an overall 5-years recurrence rate up to 78%.2-4 Especially for patients suffering from muscle-invasive tumors, high mortality is a serious health threat due to distant metastases.5,6 Chemotherapy is commonly adopted to delay relapse and prolong survival.7 However, no more than half of the muscle-invasive BC patients respond to chemotherapy.8,9 Furthermore, the problem of easy recurrence remains to be unsolved.10 Therefore, it is very important to explore novel chemotherapeutic agents with treatment and prevention functions and elucidate the mechanisms of increasing treatment responsiveness of bladder cancer cells. Inactivation of the p53 tumor suppressor pathway is a common feature of human cancers, furnishing an attractive avenue for cancer therapies based on restoring p53 function in established tumors.11,12 It has been proposed that p53 activation promoted cancer cell killing.13,14 Moreover, activation of p53 pathway can restrict malignant transformation by triggering apoptosis or cell cycle arrest.15 It is therefore highly desirable to restore p53 function by persistent p53 activation with small-molecule compounds for BC therapy. Quinazolinone is a key structural motif in many natural products, as well as in synthetic pharmaceutical compounds. Quinazolinone derivatives exhibit diverse pharmacological activities and have been identified as enzyme inhibitors,16-18 antileishmanial agents,19 receptor antagonists,20,21 anti-inflammatory agents22,23 and anticancer agents.24-26 A recent study27 aimed to identify p53 activator with potent 3



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antitumor activity documented that 5-(3-dimethylamino-propylamino)phthalazino-[1,2-b]quinazolin-8-one (BMH-727, 5ab, Figure 1) could regulate the p53 signaling pathway and exhibit anticancer activities in vitro and in vivo. However, the synthesis and further structure activity relationship studies of this scaffold have not yet been reported in the literature. In order to obtain increased efficacy, in the present study, we designed and synthesized a series of phthalazino[1,2-b]quinazolinone derivatives (5) and identified 10-bromo-5-(2-dimethylamino-ethylamino)phthalazino[1,2-b]quinazo-lin-8-one (5da, Figure 1) as the most promising one in view of both the anticancer activity and the mechanisms of action. We were able to demonstrate clearly that 5da could induce phospho-p53 mitochondria-targeted accumulation with complicated regulatory process in cell apoptosis and proliferation inhibition.

Figure 1

RESULTS Chemistry. Synthesis of phthalazino[1,2-b]quinazolinone derivatives 5 was described in Scheme 1 and Scheme 2. First, commercially available anthranilic acids were converted to the corresponding anthranilic hydrazides (2) in two steps according to literature methods.28 Then 2 and phthalic anhydride in glycol were heated in oil bath to provide 10-substituted 6H-phthalazino[1,2-b]quinazolin-5,8-diones (3) in high yield. Treatment of 3 with phosphorus oxychloride gave 10-substituted 5-chloro-phthalazino[1,2-b]quinazolin-8-ones (4) in 50-72% yields. Finally, 4 reacted with 4


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amine derivatives in toluene furnished the target compounds 5. The structure of 10-fluoro-5-(2-dimethylaminoethylamino)phthalazino[1,2-b]quinazolin-8-one

(5ba)

was further unambiguously determined by the X-ray crystallography (detailed parameters are included in the Supporting Information).

Scheme 1 Scheme 2

In Vitro Biological Evaluations. Compounds 5 were evaluated in vitro against a panel of five cancer cell lines including T24, HepG2, NCI-H460, MGC-803 and HeLa using microculture tetrazolium (MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra-zolium bromide) assay. The IC50 values derived from dose−response curves were summarized in Table 1.

Table 1

As shown in Table 1, many of the compounds showed potent cytotoxicity to all the tested cell lines. The data indicated that the substituent on the 5-position of the phthalazino[1,2-b]quinazolin-8-one skeleton had a very important effect on activity. Thirteen different substituents on the 5-position were explored. The results indicated that in general aminoalkylamine side chains could give better antiproliferative activity than 4-methyl-N-piperazinyl (series 5xe). And if the 5-substituent was replaced by a 5



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N-morpholinyl (series 5xd), alkyl (5dk) or ω-alkoxycarbonyl substituted alkyl (5dl and 5dm), the compounds almost lost the cytotoxicity. The tail of the aminoalkylamine side chain also significantly affected the antiproliferative activity. Compound with dimethylamino at the tail (5da) are more potent than with diethylamino (5df) or diiospropylamino (5dg). A tail with pyrrolidinyl and pyridyl (compounds 5dh and 5di) also result in potent antiproliferative activity. When the tail of the aminoalkylamine was replaced by a N-morpholinyl (5dc, 5dj), the cytotoxicity of the compounds would decrease. The length of the side chain also showed notable influence to cytotoxicity. A link of aminoethyl is more favorable than an aminopropyl (5da vs. 5db, 5dj vs. 5dc). The data in Table 1 also suggested that replaced the hydrogen (H) atom (5ax series) at the 10-position of the phthalazino[1,2-b]quinazolin-8-one skeleton with a halogen atom (5bx-5dx series) or a methyl (5ex series), could increase the cytotoxicity. Among them the Br substitution (5dx series) gave the most potency to most of the tested cell lines in general, as exemplified by 5da. The anticancer activities of 5da in vitro were superior to the first-line chemotherapeutic agent 10-hydroxycamptothecin (HCPT) and this compound was chosen to further investigate the mechanism of its cytotoxic effect. 5da is not Pan Assay Interference Compounds (PAINS). Prior to study the anticancer mechanism of 5da, firm experiments had been carried out to check its apparent activity was not an artifact. 5da and its analogues are known as quinoline class of putative PAINS, their absorption of light in the range 570-620 nm would 6


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interfere the effective IC50 values in turn.29 To check the potential interference effect, we tested the absorption change of light in 490 nm in T24 cells after 5da treatment. As shown in Figure 1B, 5da treatment (doses from 1 to 10 μM, 1 h) did not affect the absorption of light in 490 nm (P>0.05). Another mechanism of assay interference of such class putative PAINS may be to cause DNA damage. In order to test the possibility, DNA relaxation assay was used to investigate the interaction between 5da and DNA directly. After concentrations (1-10 M) of 5da treatment, there was nearly no difference either in conformational changes of DNA or in the mobility rate of DNA compared with the negative control group; while in the positive control group (EB group), the migration rate of DNA was significantly inhibited (Figure 1C). Moreover, a biomarker, γH2AX was adopted to detect DNA damage. As depicted in Figure 1D, no DNA damage was detected after 5da treatment. Furthermore, 5da containing a fluorophore could emit fluorescence in the range 420-450 nm (Figure 1E), thus leading to other mechanisms of assay interference. To test the possibility, T24 cells were treated with 5da (doses from 1 to 10 μM, 1 h), stained with PI and examined by FACS analysis. As shown in Figure 1F, 5da neither triggered obvious membrane disruption nor affected the distribution of cells in the FITC channel to further ensure the reliability of the data based on fluorescence analysis in the following experiments. Collectively, these results suggested that 5da is not PAINS. 5da Induces Cell Cycle Arrest and Cell Size Change in T24 cancer cells. To study the potential mechanistic pathways responsible for cell proliferation inhibition by 5da, we tested the change of cell cycle in T24 and J82 cancer cell lines. As shown 7



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in Figure 2A and D, 5da treatment (doses from 1 to 10 μM, 48 h) clearly increased the cell proportion of S/G2 phase and sub-G1 phase (a specific phase of cell death), while the cell proportions in G1 phase were markedly reduced (Figure 2B and D). Consistent with the effects on cell cycle arrest, 5da induced a more profound reduction in cell size (Figure 2C, left). The relative cell size (mean FSC-H) of 5da–treated cells were 87.7% of control cells in T24 cell line (Figure 2C, right). Collectively, these results documented the antiproliferation potency of 5da in a broad spectrum of cancer cell lines were potentially due to the S/G2 phase arrest-induced growth inhibition and cell size reduction.

Figure 2

To investigate the mechanism of 5da-induced cell cycle arrest, we firstly checked the p53 expression and p53 phosphorylation after 5da treatment, as the 5da analogue 5ab could pharmacologically activate the p53 pathway to exhibit anticancer activity.27 As shown in Figure 2E, both 5da and 5ab could induce p53 activation by promoting p53 phosphorylation in a dose-dependent manner along with little impact on p53 expression. To find out whether the anticancer effect of 5da against T24 cancer cells was due to p53 activation, we examined the anticancer activities of 5da in DU145 cancer cells (p53 mutation cell line), p53-null PC-3 cancer cells and LnCap cancer cells (wild-type p53). Our results documented that the anticancer activities of 5da in DU145 cancer cells and PC-3 cancer cells were obviously attenuated compared to that 8


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in wt-p53 LnCap cancer cells (Figure 2F). To further explore that the inhibitory effect of 5da on cell proliferation was mainly due to p53 activation, p53-siRNA was used to ablate endogenous p53. Both cells (p53 present and absent) were exposed to different concentrations of 5da for 48 h. In scRNA-treated T24 cells and control group, 5da treatment dramatically inhibited cell proliferation in a dose-dependent manner, while in p53-ablated T24 cells, the sensitivity of T24 cells to 5da was significantly decreased (Figure 2G). Furthermore, to provide more direct evidence on p53 activation, we also studied the inhibition of interactions between p53 and MDM2 after 5da treatment. As depicted in Figure 2H, both 5da and nutlin-3 effectively activated p53 in T24 cells, evident by robust increase of p21 protein. 5da was even more potent than nutlin-3, the first MDM2 inhibitor reported by Vassilev and colleagues.30 Both 5da and nutlin-3 could decrease MDM2 protein in T24 cells, but almost no effect on the expression of p53. These results suggested that 5da exhibited toxicity for cancerous was predominantly due to p53 activation. Generally speaking, p53 activation is a common response to distinct cellular stresses, which induces cell cycle arrest or apoptosis by governing the downstream signaling molecules.31 Because p21 is the direct target of p53 regulatory circuit, we investigated

the

cyclinE-CDK2

complexes,

cyclinD-CDK4

complexes

and

cyclin-dependent kinase (CDK) inhibitor p21 variance due to their functions in cell cycle progression. Cyclin E binds to CDK2, which is required for the transition from G1 to S phase during cell division.32 Our results showed that 5da could induce significant increase of cyclinE-CDK2 complexes to promote G1 to S phase transition 9



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(Figure 2I). However, nearly no change was found in the expression of cyclin D and CDK4. Instead, 5da could up-regulate p21 expression in a dose-dependent manner (Figure 2I). These results suggested that S/G2 arrest induced by 5da resulted from the inhibition of CDK activity by p21 rather than the expression reduction of cyclin D and CDK4. 5da Induces Cell Apoptosis through the Intrinsic Apoptotic Pathway. To gain further insight into the mechanisms of tumor suppression activity of 5da, we wanted to confirm firstly whether the sub-G1 phase cells induced by 5da were due to apoptosis. The 5da-treated cells were therefore investigated with PI and FITC-Annexin V staining for apoptosis identification. As depicted in Figure 3A, cells treated by 5da displayed obvious cell apoptosis in T24 cells in a dose-dependent manner. Compared to vehicle-treated cells, 5da induced 4.22% (1 μM), 26.4% (5 μM) and 29.4% (10 μM) of apoptotic cells, respectively. To confirm the proapoptotic effect of 5da in T24 cells, cell apoptosis with activated caspase-3 was also studied. As shown in Figure 3B, the peaks for cells with activated caspase-3 were gradually increased in the 5da-treated cells compared with the control group. The proportions of caspase-3 activated cells after the 5da treatment were 7.0% (1 μM), 17.6% (5 μM) and 50.0% (10 μM) in the T24 cells. Furthermore, after the treatment of 5da, Apoptosis-inducing Factor (AIF) was released from mitochondria and translocated to the nucleus to trigger apoptosis (Figure 3C). The observations prompted us to verify which death effectors in the mitochondria were dynamic participants in the death signaling, because AIF is a mitochondrial 10


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protein.33 Bcl-2 family proteins were firstly chosen to examine how the abundance of likely candidate proteins varied after 5da treatment. As shown in Figure 3D, the most increase was in the levels of Bim, Bax, and Bak, while Bcl-xl, Bcl-2 showed insignificant change. These results collectively indicated that 5da-induced cell apoptosis were mainly through mitochondrial-mediated intrinsic apoptotic pathway.

Figure 3

5da Induces Conformational Activation of Bak for Cell Apoptosis by Mitochondrial Accumulation of Phospho-p53. Conformational activation of Bak characterized by N-terminal exposure is a distinct marker of the sensitized mitochondrial apoptotic pathway.34,35 Having confirmed the activated intrinsic apoptotic pathway, we sought to find out whether 5da could conformationally activate Bak, thus defining a framework to understand the mechanism for apoptotic signals cascading. Immunohistochemistry method was adopted to examine the Bak activation. As depicted in Figure 4A, almost no activated Bak could be observed in the control group, reflecting the fact that Bak without activation could not play the role of death effector even in the case of high abundance. However, after the treatment by 5da, conformationally altered Bak could be captured by N-terminal Bak antibody, and such phenomenon was more obvious when the drug concentration increased. The results indicated that 5da could conformationally activate Bak for cell apoptosis.

11



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Conformational activation of Bak is a dissociation step from antiapoptotic Bcl-2 family members to form Bak homodimers, eventually leading to generate the pore forming activity.36 Previous studies have shown that antiapoptotic Bcl-2 family member Bcl-xl controls the conformational status of Bak by way of forming Bak–Bcl-xl complex.34,35 That fact raised the possibility that 5da-induced Bak activation may be due to disrupting the Bak–Bcl-xl interaction. To answer such a question, coimmunoprecipitation analysis was adopted to study the two proteins of interests. As expected, 5da could disrupt the Bak–Bcl-xl interaction (Figure 4C) and without affecting the expression of Bcl-xl (Figure 3D). Having established Bak activation by 5da-induced Bak–Bcl-xl complex perturbation, what interested us most was how 5da predominantly affected cells to transfer proapoptotic stimuli. 5da has been confirmed to be a p53 activator in our study. We found that 5da did not affect p53 expression, but significantly elevated p53 phosphorylation (Figure 2G), and phospho-p53 mainly accumulated in mitochondria (Figure 4B). Previous studies have shown that activated p53 always was translocated into mitochondria to trigger cell apoptosis through the transcription-independent p53 pathways.36,37 It is therefore quite reasonable for us to study the potential role of p53 in 5da-induced Bak activation. To analyze p53’s ability to perturb Bak–Bcl-xl interaction after 5da treatment, we coimmunoprecipitated phospho-p53 with Bcl-xl and Bcl-xl with Bak in T24 cells. As shown in Figure 4C, p53 exhibited direct interaction and high affinity for Bcl-xl after 5da treatment for 24 h, which was consistent with the previous report,38 while Bak dissociated from Bak–Bcl-xl complex. 12


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The results suggested that 5da-induced mitochondrial accumulation of phospho-p53 promoted Bak activation even when it stayed bound to Bcl-xl.

Figure 4

5da Shows Potential Anticancer Potency in Vivo with Low Toxicity. The potential clinical utility of 5da was finally examined in HepG2 xenograft tumors in nude mice. In the HepG2 xenograft models, mice with tumors at the volume of 80200 mm3 were randomized into treatment and vehicle control groups (n=6/group), and treated via ip injection of 5da or HCPT, respectively, every other day. The tumor-bearing mice treated by 5da (6 or 12 mg/kg/2days) showed significant tumor growth inhibition over the treatment period compared with those in vehicle-treated, tumor-bearing mice (Figure 5A, B and C). The results of tumor growth inhibition (TGI) identical to vehicle control group reached to 43.6% (5da, 6 mg/kg/2days), 61.3% (5da, 12 mg/kg/2days) and 71.6% (HCPT, 6 mg/kg/2days) respectively (P100

92.29±19.60

>100

>100

>100

5bd

>100

>100

>100

>100

>100

5cd

>100

>100

>100

>100

>100

5dd

>100

>100

>100

>100

>100

5ed

>100

>100

>100

>100

>100

5ae

33.81±6.68

39.42±8.03

31.35±5.47

44.45±7.20

50.38±9.94

5be

34.84±6.22 51.89±11.01 77.72±14.41

26.11±7.06

44.89±10.07

5ce

30.73±8.06 57.56±12.22 70.36±16.28 66.14±14.30

5de

77.02±15.28

43.70±9.07

40.39±6.71

73.5±14.33

5ee

60.48±12.38 55.19±13.61 70.56±16.64

34.18±5.19

85.00±19.88

5df

10.69±2.47

2.99±0.31

5.06±1.74

7.26±1.39

8.81±2.66

5dg

12.71±2.76

11.68±2.18

13.61±2.47

10.60±2.11

11.38±2.57

5dh

6.43±1.24

5.21±0.43

6.84±1.22

4.15±0.46

3.71±0.53

5di

3.81±0.47

4.14±0.86

7.85±2.53

4.48±0.69

2.01±0.83

5dj

10.75±1.86

11.28±2.54

7.85±1.66

9.90±1.07

8.80±1.55

5dk

>100

>100

>100

>100

>100

5dl

>100

>100

>100

>100

>100

>100

>100

>100

>100

>100

5.53±1.24

5.52±0.96

4.91±1.02

15.19±2.81

29.16±6.19

5dm b

HCPT

41.13±8.71

a

>100

Each IC50 value was calculated from 3 independent experiments performed in triplicate. b10-Hydroxycamptothecin. 46


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Figure Legends Scheme 1. Synthesis of phthalazino[1,2-b]quinazolin-8-one derivatives 5aa-5ee Scheme 2. Synthesis of phthalazino[1,2-b]quinazolin-8-one derivatives 5df-5dm

Figure 1. 5da is not Pan Assay Interference Compounds (A) Structures of 5ab and 5da. (B) Effects of 5da on absorption of light in 490 nm in T24 cells. (C) Gel electrophoresis of pBR322 DNA after being incubated with 5da at the concentrations of 1-10 μM for 1 h in Tris-HCl buffer. 1%DMSO+ plasmid in lane 1 as negative control, EB+ plasmid in lane 2 as positive control. (D) Immunoblotting analysis of γH2AX related to 5da-induced DNA damage. Whole-cell extracts were prepared and analyzed by Western blot analysis using antibody against γH2AX. (E) Emission spectra of 5da. (F) Effects of 5da on membrane disruption in T24 cells. EB: Ethidium bromide.

Figure 2. Effects of 5da on cell cycle distribution and cell size in T24 and J82 cells. (A) The regulation of cell cycle arrest by 5da was conducted by FACS analysis with PI staining in T24 cells. (B) The distribution of cell cycles in 5da-treated T24 cells was plotted. Mean ± SD was from three independent measurements. (C) 5da reduced the cell size in T24 cells. Cells were treated for 48 h with DMSO or 5 μM of 5da and analyzed by FACS for relative cell size. Histograms of the forward scatter (FSC-H) for the treated cells and mean FSC-H values were shown. (D) The regulation of cell cycle arrest by 5da was conducted by FACS analysis with PI staining in J82 cells. (E) 5da and 5ab significantly activated p53 with little influence on its expression. T24 47



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cells were incubated with indicated concentrations of 5da and 5ab for 24 h. Whole-cell extracts were prepared and analyzed by Western blot analysis using antibodies against p53 and p-p53 (s392). The same blots were stripped and reprobed with β-actin antibody to show equal protein loading. (F) IC50 values of 5da in DU145, PC-3 and LnCap cancer cells. (G) Knock down p53 for inhibition sensitivity study. T24 cells were pre-exposed to p53-siRNA or scRNA for 24 h and then treated with indicated concentrations of 5da for 48 h (H) Immunoblotting analysis of p53 activation induced by 5da. Nutlin-3 was used as a positive control. (I) Immunoblotting analysis of proteins related to 5da-induced cell cycle arrest. Whole-cell extracts were prepared and analyzed by Western blot analysis using antibodies against proteins indicated. The same blots were stripped and reprobed with β-actin antibody to show equal protein loading. **p < 0.01

Figure 3. 5da induces cell apoptosis through the intrinsic apoptotic pathway. (A) The induction of apoptosis by 5da was examined by FACS analysis with PI and FITC-Annexin V staining in T24 cells. (B) 5da induced apoptosis by triggering Caspase-3 activities in T24 cells. Arrow showed the Caspase-3-activited cells. (C) AIF translocated to the nucleus after treatment by 5da. Images were acquired using a Carl Zeiss LSM 710 microscope (magnification 400). (D) Immunoblotting analysis of proteins related to the mitochondria-mediated intrinsic apoptotic pathway evoked by 5da. Whole-cell extracts were prepared and analyzed by Western blot analysis using antibodies against proteins indicated. The same blots were stripped and 48


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reprobed with β-actin antibody to show equal protein loading.

Figure 4. 5da-induced mitochondrial accumulation of phospho-p53 conformationally activated Bak by reorganizing the Bak–Bcl-xl Complex. (A) 5da induced conformational Bak activation. Cells were immunostained with antibodies for active Bak. Images were acquired using a Carl Zeiss LSM 710 microscope (magnification 400). (B) Phospho-p53 targeting accumulation in mitochondria after treatment by 5da. T24 cells treated by 5da were subjected to subcellular fractionation, and immunoblotting was performed with cytoplasm (cytosol) and mitochondria (mito) fractions. β-Actin and cox IV were used as cytosolic and mitochondrial marker proteins, respectively. (C) Status of Bak-Bcl-xl interaction and phospho-p53-Bcl-xl interaction in T24 cells were treated with 5 μM 5da for 24 h.

Figure 5. In vivo anticancer activity of 5da in HepG2 and T24 xenograft mice. (A) The tumor volume of mice from each group during the observation period. Tumor-bearing mice were administered the vehicle (negative control), 5da (6 or 12 mg/kg per 2 days) or HCPT (6 mg/kg per 2 days, positive control). (B) The images of the excised tumors from each group. (D) Body weights of the mice recorded at the end of the treatments. (C, E) The weight of the excised tumors from HepG2 xenograft model (C) and T24 xenograft model (E). Data were presented as the mean ±SD. Error bars represented SD, n = 6, *p < 0.05 and **p < 0.01, vs control. (F) Immunoblotting analysis of p53 and Bak in vivo after 5da treatment. 49



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Scheme

Scheme 1

Scheme 2

50


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Figures

Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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52x14mm (600 x 600 DPI)


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quinazolinones as p53 Activators: Cell Cycle Arrest ... - ACS Publications

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