Damodaran Chendil

Curcumin confers radiosensitizing effect in prostate cancer cell line PC-3.

Curcumin (Diferuloylmethane) is a major chemical component of turmeric (curcuma longa) and is used as a spice to give a specific flavor and yellow color in Asian food. Curcumin exhibits growth inhibitory effects in a broad range of tumors as well as in TPA-induced skin tumors in mice. This study was undertaken to investigate the radiosensitizing effects of curcumin in p53 mutant prostate cancer cell line PC-3. Compared to cells that were irradiated alone (SF2=0.635; D0=231 cGy), curcumin at 2 and 4 μM concentrations in combination with radiation showed significant enhancement to radiation-induced clonogenic inhibition (SF2=0.224: D0=97 cGy and SF2=0.080: D0=38 cGy) and apoptosis. It has been reported that curcumin inhibits TNF-α-induced NFκB activity that is essential for Bcl-2 protein induction. In PC-3 cells, radiation upregulated TNF-α protein leading to an increase in NFκB activity resulting in the induction of Bcl-2 protein. However, curcumin in combination with radiation treated showed inhibition of TNF-α-mediated NFκB activity resulting in bcl-2 protein downregulation. Bax protein levels remained constant in these cells after radiation or curcumin plus radiation treatments. However, the downregulation of Bcl-2 and no changes in Bax protein levels in curcumin plus radiation-treated PC-3 cells, together, altered the Bcl2 : Bax ratio and this caused the enhanced radiosensitization effect. In addition, significant activation of cytochrome c and caspase-9 and -3 were observed in curcumin plus radiation treatments. Together, these mechanisms strongly suggest that the natural compound curcumin is a potent radiosesitizer, and it acts by overcoming the effects of radiation-induced prosurvival gene expression in prostate cancer.

Par-4-Dependent Apoptosis by the Dietary Compound Withaferin A in Prostate Cancer Cells

Deletion or mutation of the androgen receptor (AR) renders prostate tumors refractory to apoptosis by androgen ablation, the mainstay of prostate cancer therapy. To identify novel therapeutics that can induce apoptosis regardless of the AR status of prostate cancer cells, we screened dietary herbal compounds using a reporter assay for the prostate apoptosis response-4 (Par-4) gene, which induces p53- and PTEN-independent and cancer-selective apoptosis. One of the compounds, withaferin A (WA), a major constituent of the dietary compound Withania somnifera, induced Par-4-dependent apoptosis in androgen-refractory prostate cancer cells and regression of PC-3 xenografts in nude mice. Interestingly, restoration of wild-type AR in PC-3 (AR negative) cells abrogated both Par-4 induction and apoptosis by WA. Individually, WA and anti-androgens induced neither Par-4 nor apoptosis in androgen-responsive prostate cancer cells, yet in combination, WA and anti-androgen synergistically induced Par-4 and apoptosis in androgen-responsive prostate cancer cells. Thus, when judiciously combined with anti-androgens, WA inhibits survival of both androgen-responsive and androgen-refractory prostate cancer cells by a Par-4-dependent mechanism. As Par-4 up-regulation induces apoptosis in most tumor cells, our findings can be extended to high-throughput screens to identify synergistic combinations for both therapy-sensitive and therapy-resistant cancers.

Didox (A Novel Ribonucleotide Reductase Inhibitor) Overcomes bcl-2 Mediated Radiation Resistance in Prostate Cancer Cell Line PC-3

In this study, we investigated the influence of bcl-2 overexpression on the radiosensitizing potential of Didox (DX; 3,4-Dihydroxybenzohydroxamic acid), a novel ribonucleotide reductase inhibitor, in p53-null prostate cancer cell line PC-3. The PC-3 cells were transfected with vector alone or ectopically overexpressed with CMV-bcl-2 construct. The effect of radiation (IR) or DX alone and in combination (pre and post IR exposure of DX) on cell survival was determined by colony-forming assay. The impact of these two treatments on the cell cycle was determined by flow cytometry. To further understand the molecular mechanism of DX-mediated radiosensitization, induction of pro-survival and pro-apoptotic factors were determined by Western blot and gel-shift assays respectively. When compared to PC-3/bcl-2 cells (SF2=0.84; D0=437cGy), the PC-3/vector cells (SF2=0.4; D0=235cGy) were significantly sensitive to ionizing radiation (p

Uterine sarcomas express KIT protein but lack mutation(s) in exon 11 or 17 of c-KIT.

OBJECTIVE Several tumors express the protein product of the protooncogene c-KIT. Some of these respond to imatinib mesylate, a tyrosine kinase inhibitor. The tumors that respond frequently have mutation(s) in exon 11 of c-KIT that encodes for the regulatory juxtamembrane helix. Some tumors that express KIT protein have mutation(s) in exon 17 of c-KIT; however, these do not respond to imatinib mesylate. This investigation was performed to determine the expression of KIT protein and mutational status of exons 11 and 17 of c-KIT in uterine sarcomas. METHODS Twenty-five uterine sarcomas treated from 1990 to 2002 were evaluated. These included 14 malignant mullerian mixed tumors (MMMT), 7 leiomyosarcomas (LMS), 2 endometrial stromal sarcomas (ESS), and 2 high-grade heterologous sarcomas (HGHS). Formalin-fixed, paraffin-embedded tissue sections were immunostained with anti-KIT antibody (Santa Cruz Biotechnology, Santa Cruz, CA) with a semiquantitative assessment. Normal myometrium when present in the section was used as an internal negative control. Areas of tumor were microdissected followed by DNA extraction, polymerase chain reaction (PCR) amplification of exons 11 and 17, single-strand conformational polymorphism (SSCP), and DNA sequencing to detect the presence of mutation(s). RESULTS All 25 tumors expressed KIT protein at varying levels as assessed by immunohistochemistry. The staining was diffuse and of moderate to strong intensity in 22 tumors. In three tumors (one of each type except MMMT) the staining intensity was weak. In MMMT the epithelial and sarcomatous foci stained similarly. No mutation(s) in exons 11 or 17 of c-KIT were identified in 24/25 tumors. One LMS had deletion of both exons 11 and 17. CONCLUSIONS Although uterine sarcomas express KIT protein, they lack KIT-activating mutation(s) in exon 11 or 17 of c-KIT. Therefore, these tumors are unlikely to respond to imatinib mesylate.

Par-4, a pro-apoptotic gene, inhibits radiation-induced NF kappa B activity and Bcl-2 expression leading to induction of radiosensitivity in human prostate cancer cells PC-3.

Ionizing radiation caused induction NFκB activity and Bcl-2 protein expression in the radioresistant p53 null human prostate cancer cell line, PC-3. Exposure of PC-3 cells to Ad5-IkB super-repressor inhibited radiation-induced Bcl-2 expression indicating that radiation-induced NFκB activity is required for the induction of Bcl-2 protein. PAR-4, a novel pro-apoptotic protein is a potent down-modulator of NFκB activity and bcl-2 protein expression. This study was undertaken to investigate the impact of PAR-4 expression on radiation-induced NFκB activity and Bcl-2 expression and its resultant radiation response in PC-3 cells. Western blot analysis indicated that enforced expression of PAR-4 in PC-3 cells down regulated radiation-induced bcl-2 protein, whereas in vector transfected cells radiation caused an induction of bcl-2 protein. In both transfectant cell lines, the bax protein levels remained unaltered after radiation. When compared to PC-3/Vector cells, PC-3/PAR-4 cells showed significant sensitivity to radiation-induced clonogenic inhibition and apoptosis. Thus, the down-regulation of bcl-2 protein by ectopic PAR-4 expression altered bcl-2: bax ratio in PC-3/PAR-4 cells and this led enhanced radiosensitivity. PAR-4 was found to inhibit the radiation-induced NFκB activity and NFκB transcriptional activity is essential for bcl-2 upregulation. In PC-3/Vector cells, radiation caused an increase in NFκB activity leading to upregulation of bcl-2 protein. However, in PC-3/PAR-4 cells, the radiation-induced NFκB activity was inhibited resulting in the transrepression of bcl-2 promoter and down-modulation of bcl-2 protein. In addition, PAR-4 was found to directly inhibit the phosphorylation and degradation of IκBa, which led to the loss of NFkB activity causing repression of endogenous and radiation-induced Bcl-2 protein. Together, these mechanisms suggest that PAR-4 is functionally required to cause radiation-induced apoptosis by abrogating the survival and anti-apoptotic effects of NFκB activity and bcl-2 function respectively.

Farnesyltransferase inhibitor (L-744,832) restores TGF-β type II receptor expression and enhances radiation sensitivity in K-ras mutant pancreatic cancer cell line MIA PaCa-2

Activated ras is known to dysregulate TGF-β signaling by altering the expression of TGF-β type II receptor (RII). It is well documented that tumor cells harboring mutant ras are more resistant to radiation than cells with wild-type ras. In this study, we hypothesized that the use of farnesyltransferase inhibitor (FTI, L-744,832) may directly restore TGF-β signaling through RII expression via ras dependent or independent pathway leading to induction of radiation sensitivity. Two pancreatic cancer cell lines, BxPC-3 and MIA PaCa-2 were used in this study. FTI inhibited farnesylation of Ras protein more significantly in MIA PaCa-2 than BxPC-3 cells. In contrast, MIA PaCa-2 cells were resistant to radiation when compared to BxPC-3 cells. BxPC-3 cells were more resistant to FTI than MIA PaCa-2 cells. In combination treatment, no significant radiosensitizing effect of FTI was observed in BxPC-3 cells at 5 or 10 μM. However, in MIA PaCa-2 cells, a significant radiosensitizing effect was observed at both 5 and 10 μM concentrations (P>0.004). The TGF-β effector gene p21waf1/cip1 was elevated in combination treatment in MIA PaCa-2 but not in BxPC-3 cells. In MIA PaCa-2 cells, FTI induced TGF-β responsive promoter activity as assessed by 3TP-luciferase activity. A further induction of luciferase activity was observed in MIA PaCa-2 cells treated with radiation and FTI. Induction of TGF-β signaling by FTI was mediated through restoration of the RII expression, as demonstrated by RT–PCR analysis. In addition, re-expression of RII by FTI was associated with a decrease in DNA methyltransferase 1 (DNMT1) levels. Thus, these findings suggest that the L-744,832 treatment restores the RII expression through inhibition of DNMT1 levels causing induction of TGF-β signaling by radiation and this forms a novel molecular mechanism of radiosensitization by FTI.

Increased expression of PSA mRNA during brachytherapy in peripheral blood of patients with prostate cancer

OBJECTIVES To determine the extent of iatrogenic tumor cell dissemination during brachytherapy by assessing prostate-specific antigen (PSA) mRNA expression in circulating prostate tumor cells using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The instrumentation used in the radioisotope seed placement of the prostate causes trauma to blood vessels and provides a vascular access for tumor cells that can lead to potential iatrogenic dissemination and systemic failure. METHODS Twenty-five patients treated for brachytherapy were recruited in the study. Controls included 4 normal men and 1 woman; case controls included 4 patients who underwent prostate biopsy for prostate cancer diagnosis. Peripheral blood (10 mL) was collected before, during, and after the brachytherapy procedure. Total RNA was isolated from mononuclear cells and phosphorus-32 RT-PCR was performed to analyze the mRNA expression of PSA and G6PDH genes. RESULTS Of 25 patients, 23 were negative for PSA mRNA expression and 2 were positive for PSA mRNA expression before brachytherapy. Of the 23 patients who were negative for PSA mRNA expression before treatment, 15 patients (65%) turned positive during or after brachytherapy and the remaining 8 patients remained negative throughout the treatment. Eight of the 25 patients developed rising serum PSA levels. Of these 8 patients, 1 (12.5%) did not have PSA mRNA expression in the peripheral blood before, during, or after brachytherapy; the remaining 7 patients who developed rising serum PSA levels had PSA mRNA expression after brachytherapy (P = 0.03). CONCLUSIONS These findings strongly suggest that iatrogenic shedding of prostate cells occurs as a result of brachytherapy and raises the concern that these cells liberated at the time of brachytherapy increase the risk of metastatic deposits and results in systemic failure, as measured by serum PSA levels.

A herbal medicine for the treatment of lung cancer

Lung cancer is the leading cause of cancer-related deaths throughout the world. Extracts of medicinal plants are believed to contain different chemopreventive or chemotherapeutic compounds. In this study, we determined the anti-cancer property of one of the traditional Indian medicine Rasagenthi Lehyam (RL) for the treatment of lung cancer. Two lung cancer cell lines (A-549 and H-460) and one normal bronchial epithelial (BEAS-2B) cell line were used to test the chemotherapeutic effect of RL. Out of five fractions of RL, chloroform fraction of RL (cRL) demonstrated a significant inhibition of cell proliferation and induction of apoptosis in A-549 and H-460 cells but not in normal BEAS-2B cells. The cRL fraction up-regulated the pro-apoptotic genes p53 and Bax and induced caspase-3 activation, and down-regulated the pro-survival gene Bcl-2 in both the lung cancer cell lines. Also, nuclear export of p53 was seen in cRL-treated lung cancer cells. In addition, cRL induced G2/M arrest of cell cycle and enhanced the radio-sensitivity of both the lung cancer cell lines. This study suggests that cRL may prove to be a potent anti-cancer agent that may be used for the treatment of lung cancer. However, further studies are required to bring cRL into the mainstream of medicine in the treatment of lung cancer. (Mol Cell Biochem xxx: 125–133, 2005)

The radiosensitization effect of 2-deoxy-D-glucose on human glioma cells.

Commentary to: Differential Mechanisms of Radiosensitization by 2-deoxy-D-glucose in the Monolayers and Multicellular Spheroids of a Human Glioma Cell Line Divya Khaitan, Sudhir Chandna, M.B. Arya and B.S. Dwarakanath