Priya Srinivas

Emodin induces apoptosis of human cervical cancer cells through poly(ADP-ribose) polymerase cleavage and activation of caspase-9

Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an active herbal component traditionally used in China for treating various ailments. Emodin exerts antiproliferative effects in many cancer cell lines and the actual molecular mechanism of which is still not clear. Since apoptosis could be a potential mechanism to explain these effects, we tested whether emodin induces cell death in human cervical cancer cells. Our results suggest that emodin exerts antiproliferative effects in human cervical cancer cells. Emodin inhibited DNA synthesis and induced apoptosis as demonstrated by increased nuclear condensation, annexin binding and DNA fragmentation in Bu 25TK cells in the presence of emodin. Moreover, we demonstrate for the first time in human cervical cancer cells that the apoptotic pathway involved in emodin-induced apoptosis is caspase-dependent and presumably through the mitochondrial pathway, as shown by the activation of caspases-3, -9 and cleavage of poly(ADP-ribose) polymerase.

Plumbagin induces reactive oxygen species, which mediate apoptosis in human cervical cancer cells

There is an emerging evidence that plumbagin (5‐hydroxy‐2‐methyl‐1, 4‐naphthoquinone) may have potential as a chemotherapeutic agent. However, the growth inhibitory mechanisms of plumbagin have remained unexplored. The aim of the study was to determine whether plumbagin‐induced cell death in human cervical cancer cell line, ME‐180, exhibited biochemical characteristics of apoptosis and to check whether N‐acetyl‐l‐cysteine (NAC), which is a free radical scavenger, can reverse the cytotoxic effects of plumbagin. It can be concluded from the results that plumbagin inhibits the growth of ME‐180 cells in a concentration and time‐dependent manner. The cytotoxic effect of plumbagin induced cell death is through the generation of reactive oxygen species (ROS) and subsequent induction of apoptosis as demonstrated by the present data. Treatment of cells with plumbagin caused loss of mitochondrial membrane potential (ΔΨm), and morphological changes characteristic of apoptosis, such as the translocation of phosphatidyl serine, nuclear condensation, and DNA fragmentation. Moreover, plumbagin‐induced apoptosis involved release of mitochondrial cytochrome c and apoptosis inducing factor (AIF), thus activation of caspase‐dependent and ‐independent pathways, as shown by the plumbagin‐mediated activation of caspase‐3 and ‐9. Our results also show that pretreatment of ME‐180 cells with NAC blocks plumbagin‐induced loss of ΔΨm and subsequent release of cytochrome c, AIF, and caspase‐9 and ‐3 activation, thus inhibiting the apoptotic ability of plumbagin.

Nimbolide retards tumor cell migration, invasion, and angiogenesis by downregulating MMP-2/9 expression via inhibiting ERK1/2 and reducing DNA-binding activity of NF-κB in colon cancer cells.

Nimbolide, a plant‐derived limonoid has been shown to exert its antiproliferative effects in various cell lines. We demonstrate that nimbolide effectively inhibited proliferation of WiDr colon cancer cells through inhibition of cyclin A leading to S phase arrest. It also caused activation of caspase‐mediated apoptosis through the inhibition of ERK1/2 and activation of p38 and JNK1/2. Further nimbolide effectively retarded tumor cell migration and invasion through inhibition of metalloproteinase‐2/9 (MMP‐2/9) expression, both at the mRNA and protein level. It was also a strong inhibitor of VEGF expression, promoter activity, and in vitro angiogenesis. Finally, nimbolide suppressed the nuclear translocation of p65/p50 and DNA binding of NF‐κB, which is an important transcription factor for controlling MMP‐2/9 and VEGF gene expression.

Radiosensitizing effects of plumbagin in cervical cancer cells is through modulation of apoptotic pathway

Radiotherapy is the primary line of cancer treatment for cervical cancer and is known to induce cell death in tumors. Radiotherapy is however limited by the total dose that can be given without damaging normal tissue. Plumbagin, a naturally occurring naphthaquinone, has been reported to have free radical producing properties. Hence we hypothesized that plumbagin could also have properties that could modify effects of radiation on cervical cancer cells. Radiation in combination with plumbagin may thus have treatment augmenting effects. Results from our studies have shown that a lower dose of radiation in combination with plumbagin could induce apoptosis more effectively compared to a higher dose of radiation alone. Plumbagin in combination with 2 Gy of radiation was very effective in inducing apoptosis, when compared to a higher radiation dose of 10 Gy alone. This combination also showed a fivefold increase in the activation of caspase 3 in C33A cells. Activation of effector caspases confirms that the induction of apoptosis by irradiation and plumbagin involves caspase‐dependent pathways. Expression of apoptotic regulatory molecules Bcl‐2, Bax and Survivin was also modulated by plumbagin in combination with radiation. In summary, this study shows that a combination of plumbagin and radiation augmented cell growth inhibition compared to higher radiation dose alone, thus indicating that plumbagin may be a potential radiosensitizer acting through the induction of apoptosis.

Aloe emodin inhibits colon cancer cell migration/angiogenesis by downregulating MMP-2/9, RhoB and VEGF via reduced DNA binding activity of NF-κB.

Aloe emodin (AE), a natural anthraquinone, is reported to have antiproliferative activity in various cancer cell lines. In this study we analyzed molecular mechanisms involved in the antimigratory and antiangiogenic activity of this hydroxy anthraquinone in colon cancer cell, WiDr. Our results show that a relatively non toxic concentration of AE suppressed the phorbol-12-myristyl-13-acetate (PMA) induced migration and invasion of tumor cells. On analysis for the molecules involved in the migration/invasion, we found AE downregulated mRNA expression and promoter/gelatinolytic activity of Matrix Metalloproteinase (MMP)-2/9, as well as the RhoB expression at gene and protein level. It was also a strong inhibitor of Vascular Endothelial Growth Factor (VEGF) expression, promoter activity and endothelial cell migration/invasion and in vitro angiogenesis. AE suppressed the nuclear translocation and DNA binding of NF-κB, which is an important transcription factor for controlling MMP-2/9 and VEGF gene expression. Taken together these data indicate that AE target multiple molecules responsible for cellular invasion, migration and angiogenesis. Inhibitory effect on angiogenic and metastatic regulatory processes make AE a sensible candidate as a specific blocker of tumor associated events.

Insidious role of nitric oxide in migration/invasion of colon cancer cells by upregulating MMP-2/9 via activation of cGMP-PKG-ERK signaling pathways.

Nitric oxide (NO), an uncharged free radical is implicated in various physiological and pathological processes. The present study is an investigation on the effect of NO on proliferation, apoptosis and migration of colon cancer cells. Colon adenocarcinoma cells, WiDr, were used for the in vitro experiments. Tissues from colon adenocarcinoma, adjacent normal and inflammatory tissue and lymph node with metastasis were evaluated for iNOS, MMP-2/9 and Fra-1/Fra-2. NO increases the proliferation of cancer cells and simultaneously prevents apoptosis. Expression of MMP-2/9, RhoB and Rac-1 was enhanced by NO in a time dependent manner. Further, NO increased phosphorylation of ERK1/2 and induced nuclear translocation of Fra-1 and Fra-2. Electrophoretic mobility shift analysis and use of deletion mutant promoter constructs identified role of AP-1 in NO-mediated regulation of MMP-2/9. iNOS, MMP-2/9, Fra-1 and Fra-2 in normal and colon adenocarcinoma tissues were analyzed and it was found that increased expression of these proteins in cancer when compared to normal provides support to our in vitro findings. The study showed that the NO-cGMP-PKG promotes MMP-2/9 expression by activating ERK-1/2 and AP-1. This study reveals the insidious role of NO in imparting tumor aggressiveness.

Antisense blocking of BRCA1 enhances sensitivity to plumbagin but not tamoxifen in BG-1 ovarian cancer cells

Previous studies have shown that reduction in BRCA1 mRNA and protein can result in increased proliferation of BG‐1 ovarian cancer cells in both in vitro and in vivo conditions, suggesting that BRCA1 may normally act as a growth inhibitor in these cells. Also, there are other reports that suggest that wild‐type BRCA1 protein may repress estrogen receptor (ER) function either directly or indirectly. However, response to antiestrogen drugs in BRCA1‐blocked ER‐positive ovarian cancer cells has not been reported, and this served as the rationale for this study. We analyzed the effect of tamoxifen, emodin, and plumbagin in BRCA1‐blocked ER‐positive BG‐1 ovarian cancer cells. For all three drugs, BRCA1‐blocked cells were more sensitive than the corresponding control cells as assessed by MTT assay; however, only plumbagin showed a statistically significant difference in mean viability (P < 0.05). All three drugs induced loss of mitochondrial membrane potential (ΔΨm), nuclear condensation, DNA fragmentation, and morphological changes, as observed after 6 h of drug treatment, suggesting apoptosis induction in both BRCA1‐blocked and control cells. However, apoptosis induction was greater in BRCA1‐blocked cells, the efficacy being in the order of plumbagin > tamoxifen > emodin. The dose of plumbagin needed to kill 50% was 5 μM in the control cells and 2.68 μM for the BRCA1‐blocked cells, indicating that the latter was about twofold more sensitive to plumbagin than the wild‐type cells. This throws light on the fact that plumbagin may have chemotherapeutic potential as an anticancer agent in BRCA1‐mutated ovarian cancer patients.

Aloe Emodin Induces G2/M Cell Cycle Arrest and Apoptosis via Activation of Caspase-6 in Human Colon Cancer Cells

Aloe emodin (AE), a natural anthraquinone, is reported to have antiproliferative activity in various cancer cell lines. In this study, we analyzed the molecular mechanisms involved in the growth-inhibitory activity of this hydroxyanthraquinone in colon cancer cell, WiDr. In our observation AE inhibited cell proliferation by arresting the cell cycle at the G2/M phase and inhibiting cyclin B1. AE appreciably induced cell death specifically through the induction of apoptosis and by activating caspases 9/6. Apoptotic execution was found to be solely dependent on caspase-6 rather than caspase-3 or caspase-7. This is the first study indicating that the AE induces apoptosis specifically through the activation of caspase-6.

The molecular response of vanadium complexes of nicotinoyl hydrazone in cervical cancers--a possible interference with HPV oncogenic markers.

AIMS Hydrazones belonging to the class of NNO donor Schiff bases are reported to have extensive anti-viral activity and anti-neoplastic activity against certain cancers such as colon cancer, hepatocellular carcinoma and testicular cancer. Here we aim to study the possible effects of two novel nicotinoyl hydrazones on Human papillomavirus (HPV) infected cervical cancers. MAIN METHODS The effect of vanadium complexes on the proliferation of SiHa and HeLa cells was analyzed using MTT assay. The apoptotic potentials of the complexes were assessed by their ability to induce DNA condensation as well as loss of mitochondrial membrane potential. Caspase activity assay and DNA content analysis were performed to understand the mechanism of apoptotic induction. RT-PCR analysis of cell cycle genes, GADD45, p53, p21 and HPV specific oncogenes, E6 and E7 were used to elucidate the molecular mechanism of the complexes. KEY FINDINGS OVK 49 exhibits an increased apoptosis inducing potential when compared to OVK 89 in HPV16 positive SiHa cells compared to HPV18 positive HeLa. A down regulation for E6 and E7 mRNA transcripts along with the induction of p53 protein in SiHa cells were observed when treated with OVK 49 indicating that OVK 49 might have promising anti-cancer activity against HPV16 positive cervical cancers. SIGNIFICANCE This is the first study demonstrating that vanadium complexes could induce a p53 dependent apoptotic mechanism in high risk HPV16-positive cervical cancers.

Structure activity relationship of plumbagin in BRCA1 related cancer cells

It has been shown earlier that plumbagin, a naturally occurring naphthaquinone has specific anticancer activity in BRCA1 blocked ovarian cancer cells. Plumbagin can induce estrogen dependent cell signaling and apoptosis in BRCA1 blocked ovarian cancer cells. Being a reactive oxygen species (ROS) generator and apoptosis inducing agent, plumbagin has immense potential as a promising anticancer agent. In this study we analyzed whether there would be increased anticancer activity if the positions of the functional groups on plumbagin were altered and further to analyze the detailed molecular mechanism of action of the lead molecule. Methods like MTT assay, apoptosis analysis by flow cytometry, assessment of mitochondrial membrane potential‐Δψm, suppression subtractive hybridization, microarray, molecular docking and estrogen receptor–DNA binding activity by electrophoresis mobility shift assay (EMSA) were adopted for assessing the anticancer activity. Consequently we found that, plumbagin was the most potent anticancer agent when compared to structurally related compounds. The anti‐cancer activities were in the order plumbagin > 1,4‐naphthaquinone > juglone > lawsone > menadione. Molecular docking studies showed that plumbagin could be well docked in the receptor ligand complex of TRAIL–DR5 complexes to activate the extrinsic pathway of apoptosis. Since the antiproliferative activity of plumbagin could be reduced by inhibiting ERα, we speculated that plumbagin interferes with the binding of ERα to ERE and we confirmed this by EMSA. This study clearly indicates that plumbagin can induce multiple pathways of apoptosis and cell cycle arrest in BRCA1 blocked cells compared to unblocked cells.