Design, synthesis and biological evaluation of selective survivin inhibitors

Abstract

The differential distribution between cancer cells and normal adult tissues makes survivin a very attractive cancer drug target. We have previously reported a series of novel selective survivin inhibitors with the most potent compound MX106 reaching nanomolar activity in several cancer cell lines. Further optimization of the MX106 scaffold leads to the discovery of more potent and more selective survivin inhibitors. Various structural modifications were synthesized and their anticancer activities were evaluated to determine the structure activity relationships for this MX106 scaffold. In vitro anti-proliferative assays using two human melanoma cell lines showed that several new analogs have improved potency compared to MX106. Very interestingly, these new analogs generally showed significantly higher potency against P-glycoprotein overexpressed cells compared with the corresponding parental cells, suggesting that these compounds may strongly sensitize tumors that have high expressions of the P-glycoprotein drug efflux pumps. Western blotting analysis confirmed that the new MX106 analogs maintained their mechanism of actions by selectively suppressing survivin expression level among major inhibitors of apoptotic proteins and induced strong apoptosis in melanoma tumor cells.

Keywords

selective survivin inhibitors / structure activity relationships / melanoma / human epidermoid carcinoma / colorectal cancer / P-glycoprotein drug efflux pumps

1 Introduction

Prostate cancer is the second most common malignant tumor in men in Western countries, and the number of newly diagnosed prostate cancer patients is 129.4 per 100,000 men per year^[]. Among various risk factors for prostate cancer, genetic factors, including race, family, and specific gene variants, might be associated with prostate cancer. Especially, among specific gene variants, mutations inBRCA1 and BRCA2, Hereditary Prostate cancer gene 1 (HPC1), and TMPRSS2-ERG gene fusion are linked to prostate cancer^[-^].

Recently, several studies have reported that the genetic polymorphisms of several genes at 17q12, 17q24.3, and 8q24 are significantly associated with prostate cancer^[–^]. There is growing interest in the role of genetic polymorphism in the risk for prostate cancer according to genetic polymorphism in patients with prostate cancer.

Prostate stem cell antigen (PSCA) is a cell-membrane glycoprotein consisting of 123 amino acids and highly expressed in the prostate and bladder^[,^]. Furthermore, PSCA is highly expressed in human prostate cancer, but has limited expression in normal tissues, which has become a potential target biomarker in prostate cancer. Interestingly, Jounget al.^[] have demonstrated that men with the rs1045531AA genotype ofPSCA are at high risk of prostate cancer in genotype analysis. However, there have been few reports about the relationship between rs2294008 ofPSCA and prostate cancer. Therefore, we evaluated the association between rs2294008 and the risk of prostate cancer.

2 Materials and methods

2.1 Study population

This study recruited data from 240 patients who underwent radical prostatectomy or palliative transurethral resection of the prostate (TURP) and were histologically confirmed to have adenocarcinoma of the prostate and 306 benign prostatic hyperplasia (BPH) patients for controls. Gleason scores (GS) were assigned to 12 specimens obtained from core transurethral biopsy, TURP, or radical prostatectomy. Tumor stage was estimated for specimens obtained from radical prostatectomy, and TNM 2002 stage was determined using computed tomography (CT), magnetic resonance imaging, or bone scanning.

Collection and analysis of the samples were approved by the Institutional Review Board of Chungbuk National University, and each subject provided written informed consent (IRB approval number: 2010-12-010).

2.2 Blood sampling, DNA extraction, and genotype assays of polymorphism

From each patient, a 5 mL of blood sample of was collected with a 0.1 mL EDTA tube before the operation, frozen in liquid nitrogen, and stored at –80°C. Genomic DNA was extracted from human whole blood for genotyping using a genomic DNA purification kit (Promega, Madison, WI, USA) in accordance with the manufacturer's instructions.

PSCA genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The PCR primers for PSCA rs2294008 were 5′-AAACCCGCTG- GTGTTGACT-3′ (sense) and 5′-TCCCTTCCTCCTTCCTTCC-3′ (antisense). DNA (200 ng) was amplified in a total volume of 20 µL, containing 10 pmol of each primer, 0.5 UTaq polymerase, 2.5 mmol/L deoxyribonucleotide triphosphate, and 10xPCR buffer. PCR conditions were as follows: initial denaturation step at 95°C for 5 minutes and then 39 cycles of amplification were carried out at 95°C for 45 seconds, 50°C for 45 seconds, and 72°C for 60 seconds, with a final extension step at 72°C for 10 minutes. PCR products were digested overnight at 37°C withNla III (New England BioLabs, Beverly, MA, USA) according to the manufacturer's instructions and then separated by electrophoresis on 3% agarose with ethidium bromide staining. PCR products were shown to be digested into 3 types of TT (98 and 158 bp), CT (98, 158, and 256 bp), and CC (256 bp) genotypes.

2.3 Statistical analysis

Clinical variables, such as age, GS, PSA, stage, and metastasis, according to genetic polymorphism were compared by the Chi-square test. Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated using logistic regression models. Wild-type genotypes (CC) were considered baseline risk. Using the Hardy–Weinberg equilibrium test, the expected frequency of control genotypes was analyzed. Statistical analyses were performed by using Statistical Package for Social Sciences version 23.0 (IBM, Armonk, NY, USA). All tests were performed using 2-tailed analysis and aP value of<0.05 was considered statistically significant.

3 Results

3.1 Baseline characteristics

The mean age of 240 prostate cancer patients was 68.58±7.12 years, and that of 306 controls was 70.12±7.50 years. One hundred and fourteen (47.5%) patients had stage T2, 80 (33.3%) had stage T3, and 46 (19.2%) had stage T4 prostate cancer. The baseline characteristics of the patients are summarized inTable 1.

Tab.1 Baseline characteristics of the subjects

†Prostate cancer compared with controls, P<0.05.

3.2 Association between the risk of prostate cancer and PSCA (rs2294008)

Table 2 shows genotypes and allele frequencies for rs2294008 in prostate cancer patients and controls. For prostate cancer, the CC, CT, and TT genotypes were observed in 22.5%, 56.2%, and 21.3% of the patients, respectively, and for controls, 25.5%, 53.3%, and 21.2%, respectively. The genotype frequencies for rs2294008 followed the Hardy–Weinberg equilibrium (P= 0.623). Compared with the CC genotype, the CT and TT genotypes were associated with insignificant risk of prostate cancer (P= 0.627 and 0.397, respectively).

Tab.2 Association between the risk of prostate cancer and the PSCA gene rs2294008

†Genotype distribution and Hardy-Weinberg equilibrium (P=0.623). OR: odds ratio; CI: confidence interval

3.3 Relationship between PSCA and clinicopathologic parameters in prostate cancer

As shown in Table 3, there was no significant difference in rs2294008 according to clinicopathologic parameters, such as age, GS, PSA, stage, and metastasis in prostate cancer (P>0.05 for each).

Tab.3 Relationship between PSCA (rs2294008) and clinicopathological parameters in prostate cancer

3.4 Stratified analysis of PSCA according to prostate cancer classification

The association between PSCA and prostate cancer was further evaluated by stratified analysis of age, GS, PSA, pathologic stage, and metastasis. As shown inTable 4, age, GS, PSA, pathologic stage, and metastasis did not modify the association betweenPSCA and the risk of prostate cancer (P>0.05 for each).

Tab.4 Stratified analysis of the PSCA gene (rs2294008) according to prostate cancer classification

OR: odds ratio; CI: confidence interval

4 Discussion

In this study, we investigated the role of the genetic variation of PSCA rs2294008 in prostate cancer susceptibility. It was found that the variation of PSCA rs2294008 was not associated with prostate cancer.

PSCA was initially identified and isolated as a tumor antigen highly expressed in prostate cancer^[]. PSCA plays a role in signal transduction and cell-growth regulation^[]. PSCA is highly expressed in the prostate, bladder, and pancreatic carcinomas, but downregulated in gastric, esophageal, and head-and-neck carcinomas^[,–^]. Therefore, the role of PSCA in carcinogenesis remains still controversial.

The genetic polymorphism of PSCA (rs2294008) is associated with the increased incidence of gastric cancer in the Korean population^[]. Songet al.^[] have reported that the CT and TT genotypes are associated with a significantly increased risk of gastric cancer (OR= 1.5 and 1.71, respectively) compared to the CC genotype. However, Luet al.^[] have demonstrated that rs2294008 variant genotypes (CC/CT) are not associated with increased risk of gastric cancer in the Chinese population. Thus, the genetic polymorphism ofPSCA and its association with gastric cancer risk may differ among races. Similarly, in bladder cancer, Yateset al.^[] have suggested that genetic polymorphism on 8q24 is associated with an increased risk of bladder cancer, but not associated with disease aggressiveness. In addition, Fuet al.^[] have indicated that rs2294008 is important for bladder cancer susceptibility.

Analysis of 12 genotypes for PSCA revealed that the rs1045531 is significantly associated with an increased risk of prostate cancer and that CCCAGGTACGG and CGA haplotype carriers have a significant association with risk of prostate cancer^[]. To the best of our knowledge, there is only one study by Jounget al. [()] on the association between prostate cancer risk and genetic polymorphisms ofPSCA.

There are some limitations in the present study. The sample size of our study is relatively small for identifying genetic susceptibility. We investigated only thePSCA gene rs2294008 among various PSCA genetic polymorphisms. Further large cohort studies are needed to confirm our results. Also, large cohort studies on genetic variants ofPSCA in Western countries are required to identify the association between the risk of prostate cancer and SNP.

In conclusion, the genetic polymorphism of PSCA rs2294008 was not associated with the risk of prostate cancer. Our results suggest that rs2294008 may not play a role in the pathogenesis of prostate cancer. However, sincePSCA as a transmembrane protein is associated with prostate cancer, additional studies on polymorphisms in thePSCA gene in addition to rs2294008 are needed to confirm the association between PSCA SNP and prostate cancer.

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Acknowledgment

This work supported by NIH/NCI grant 1R01CA 193609-01A1. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH/NCI. We thank Dr. Susan E Bates (Columbia University) for the SW620, SW620/Ad300 cell lines. We thank Dr. Shin-Ichi Akiyama (Kagoshima University, Japan) for the KB-3-1 and KB-C2 cell lines.

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