Prostate cancer is the leading cause of cancer-related death in men in the United States. A major cause of drug resistance in prostate and other epithelial tumors may be due to the presence of a fraction of tumor cells that retain the ability to initiate tumors and hence are termed tumor-initiating cells (TIC) or cancer stem cells. Here, we report that darinaparsin, an organic derivative of arsenic trioxide, is cytotoxic to prostate cancer cell lines as well as fresh prostate cancer cells from patients at low micromolar concentrations, and importantly inhibits the TIC subpopulations. It also inhibits growth of the castrate-resistant Du145 prostate tumor propagated as xenograft in mice and inhibits the tumorinitiating potential of prostate cancer cells. Although the mechanism by which darinaparsin acts is not completely known, we show that it kills prostate cancer cells by blocking cells in the G 2 -M phase of the cell cycle and inhibits Hedgehog signaling by downregulating Gli-2 transcriptional activity. These data provide a rationale for evaluating darinaparsin in patients with castrateresistant prostate cancer.
BACKGROUND. Prostatic small cell carcinoma is increasing in frequency in patients resistant to all forms of androgen ablation. It is a high-grade malignant neoplasm with neuroendocrine features. There are no suitable cellular models to study this particular type of carcinoma. A recent study showed that the PC-3 cell line has neuroendocrine characteristics (1). This report prompted us to determine if this cell line is enriched in tumor initiating cells, and to use this cell line as a model to determine sensitivity to second generation anti androgen treatments and chemotherapeutic agents. METHODS. To determine the percentage of tumor initiating cells (TICs) marker expression comparing LNCaP with PC-3 cells, flow cytometry analysis was used. Functional assays such as migration, invasion, colony formation and sphere formation were used to determine metastatic potential. Secondary and tertiary spheroid assays were used to determine the self-renewal ability. The CellTiter 96® AQueous non-radioactive cell proliferation assay was used to identify response to hormone deprivation and chemotherapeutic drugs. RESULTS. The growth rate of PC-3 was similar to LNCaP cells while the TIC marker CD44 was only expressed on PC-3 cells. Both LNCaP and PC-3 cells expressed α2β1 with the highest expression detected in PC-3 cells. No expression of CD133 was detected in either of the cell lines. PC-3, as compared to LNCaP cells, displayed an increase in 5-minute collagen-I attachment, cell migration, invasion, colony formation and sphere formation in an anchorage-independent environment. PC-3 cells were capable of self-renewal in the secondary and tertiary spheroid assays and expressed neuroendocrine markers such as chromogranin A and EMT marker, Twist. PC-3 cells were able to also grow in androgen-deprived media, and as compared to LNCaP cells, demonstrated resistance to anti-androgen drugs that included enzalutamide and abiraterone and chemotherapy drugs such as doxorubicin and taxotere. Surprisingly, PC-3 cells were more sensitive to methotrexate than LNCaP or DU145 cells. PC3 cells had lower levels of MTAP (methylthioadenosine phosphorylase) and hence may be the reason for their sensitivity to denovo purine synthesis inhibitors such as methotrexate. CONCLUSIONS. The novel findings presented here provide evidence that castrate resistant PC-3 cells are CD44+/ α2β1+/CD24low/CD133_ and possess stem cell properties and may serve as a useful model to study the role of new treatments for prostate cancer with neuroendocrine features. Reference: 1) Prostate. 2011 Nov; 71(15): 1668-79; PC3 is a cell line characteristic of prostatic small cell carcinoma; Tai S, Sun Y, Squires JM, Zhang H, Oh WK, Liang CZ, Huang J Citation Format: Nitu Bansal, Lisa Wu, Nadine Johnson Farley, Philip Tedeschi, Joseph R. Bertino. The castrate resistant PC-3 cell line with neuroendocrine features is enriched in tumor initiating cells and is resistant to enzalutamide and abiraterone but sensitive to antimetabolites. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4208. doi:10.1158/1538-7445.AM2015-4208
BACKGROUND: Mutation or inactivation of the retinoblastoma protein is frequently involved in prostate cancer tumorigenesis resulting in overexpression/deregulation of E2F activity. E2F1-3a overexpression induces genes involved in DNA synthesis and leads to abnormal cellular proliferation, tumor growth, and invasion. Therefore, inhibiting the overexpression of one or more activating E2Fs is a recognized target in cancer therapeutics. In our previous studies we showed that a novel penetratin conjugated 7-mer peptide (PEP) bound tightly to an immobilized consensus E2F1 promoter sequence, was cytotoxic at low micro molar concentrations to many malignant cell lines and as the PEP was unstable in serum, the PEP was encapsulated in PEGylated liposomes and treatment of tumor xenografts of small cell lung cancer H-69 and DU145 tumors propagated in mice caused tumor regression. OBJECTIVE: To determine the antitumor activity and stability of two different modified penetratin peptides: D-Arg PEP (substituting L-Arginine with D-Arginine in the peptide sequence) and N-acetylated as well as C-methylated PEP analog. METHODS: DU145 (prostate cancer) and H196 (small cell lung cancer) cells were used. To compare the efficacy of the peptides, we tested the IC50s of peptides at different time points using the MTS assay. Drug combination experiment results were analyzed using the combination index (CI) method. Peptide conformational studies were carried out using the Amber 12 suite of biomolecular simulation programs. RESULTS: Molecular simulation studies showed that the D-Arg PEP secondary structure is more stable than the L-Arg peptide structure in water. D-Arg PEP was more potent compared to L-Arg PEP, and it was also found to be more resistant to degradation by serum proteases than the L-form. The other modified form, N-acetylated, C-methylated PEP was marginally more effective than the unmodified PEP. Drug combination studies showed that the D-Arg PEP in combination with docetaxel, caused synergistic cytotoxicity against DU 145 cells. Our findings validate D-Arg peptide, an inhibitor of E2F1and 3a transcription, as a drug candidate for targeted molecular therapy of prostate cancers with elevated levels of activated E2F’s. Studies in progress are evaluating the combination of the PEGylated liposome encapsulated D-Arg PEP in combination with docetaxel against DU145 xenografts and against primary prostate cancer cells. Supported in part by a grant from the Lung Cancer Research Foundation. Citation Format: Tazeem Shaik, Nitu Bansal, Nadine Johnson Farley, John Kerrigan, Olga Garbuzenko, Tamara Minko, Emine Abali, Zoltan Szekely, Kathleen Scotto, Debabrata Banerjee, Joseph Bertino. Antitumor studies of an E2f1 promoter sequence binding peptide - penetratin conjugate as a molecule targeting E2f in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1237. doi:10.1158/1538-7445.AM2015-1237
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