Gopal 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.

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.

Estrogen-dependent cell signaling and apoptosis in BRCA1-blocked BG1 ovarian cancer cells in response to plumbagin and other chemotherapeutic agents

BACKGROUND Cellular response to chemotherapeutic drugs in the absence of BRCA1 either completely or partially had drawn less attention. The present study evaluated whether there is a differential inhibition of cell growth by selected compounds with respect to BRCA1 status in estrogen receptor (ER)-positive ovarian cancer cells. MATERIALS AND METHODS The BG1 ovarian cancer cells used in the experiments were antisensely blocked with BRCA1 gene. Growth inhibition and apoptotic induction were analyzed to evaluate the cytotoxic effects. Small interfering RNA (SiRNA) transfection, western blot analysis, RT-PCR analysis and molecular modeling were carried out to analyze the estrogen-dependent action of plumbagin. RESULTS Although we found that all the compounds studied induce apoptosis, the induction was in the order of plumbagin > doxorubicin > tamoxifen > cisplatin. Plumbagin can bind to the active site of ER-alpha. Plumbagin, however, induced ER-alpha 46 kDa truncated isoform, which was found abundantly preempted in the cytoplasm compared with a 66-kDa full-length isoform. The truncated isoform is known to inhibit classical ER-alpha signaling pathways. SiRNA-transfected cells for ER-alpha exhibited lower cytotoxicity upon plumbagin treatment than the control-transfected cells. CONCLUSION Taken together, this study indicates that plumbagin has chemotherapeutic potential in BRCA1-mutated/defective ER-positive cancers.

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.

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.

Isolation of ckit-Positive Cardiosphere-Forming Cells from Human Atrial Biopsy

There is increasing interest in developing cell-based therapies to regenerate functional muscle and blood vessels in infarcted dysfunctional myocardium, using stem cells resident in the adult heart. The objective of our study was to identify an easy and cost-effective method for the isolation and expansion of human adult cardiac-resident stem cells. The cells were isolated from right atrial biopsy samples obtained from patients with ischemic heart disease, who were undergoing coronary artery bypass grafting. Two different isolation methods, enzymatic and nonenzymatic, were employed. The cell yield and cluster formation were not significantly different with either of the techniques used for cell isolation. The nonenzymatic method is recommended because of its simplicity and lower cost compared to the enzymatic method.

IN VITRO DRUG SENSITIVITY AND APOPTOSIS INDUCTION IN NEWLY DIAGNOSED PEDIATRIC ACUTE LYMPHOBLASTIC LEUKEMIA: Correlation with Overall Survival

The present study looked for any associations between in vitro drug sensitivity and clinical outcome in pediatric acute lymphoblastic leukemia (ALL) with the standard drugs used for leukemia therapy. A total of 72 samples were analyzed. In vitro sensitivity to drugs was tested by a methyl-thiazol-tetrazolium assay in 6 serial fold dilutions. Apoptosis was determined by TUNEL assay and apoptotic index was calculated for each sample. Patients sensitive to prednisone, asparginase, vincristine, and 6-mercapto purine had higher overall survival compared to patients whose tumor cells were resistant to these drugs (p <. 01). For the other drugs tested, overall survival did not vary from that of the resistant patients. For doxorubicin, asparginase, vincristine, prednisone combination sensitivity, there was a significant worsening of prognosis from the extremely sensitive patients through an intermediate sensitive group to a most resistant group. The present study thus shows that in vitro drug-sensitivity testing provides significant prognostic information in childhood ALL.