Pranjal Sarma

Interaction Map and Selection of microRNA Targets in Parkinson's Disease-Related Genes

Parkinsons disease (PD) is a complex multigenic neurodisorder frequently occurring in elderly persons. To investigate noncoding tiny microRNA mediated gene regulation, miRanda (version 1.0b) was used to predict human miRNA target sites on selected 29 genes related to PD. To verify output generated from miRanda, a similar analysis was performed only for microRNA target sites in 3′UTR using TargetScan (version 5.1). Data extracted by miRanda elucidates the mode of microRNA action based on the location of target sites in the Parkinson genes. Sites prone to action of multiple miRNAs were identified as “hot spots.” Important properties of each miRNA including multiplicity and cooperativity appear to contribute towards a complex interplay between miRNAs and their targets. Two sets of predicted results were explored for the occurrence of target sites of 112 miRNAs expressed in midbrain. Overall, convergence of results predicted by two algorithms revealed that 48 target sites for midbrain-specific miRNA occur in close proximity in 9 genes. This study will pave a way for selection of potential miRNA candidates for Parkinsons disease-related genes for quick therapeutic applications and diagnosis.

Synthesis and biological evaluation of 4β-acrylamidopodophyllotoxin congeners as DNA damaging agents

A series of new 4β-acrylamidopodophyllotoxin derivatives (13a-o) were synthesized by coupling of substituted acrylic acids (10a-l and 11m-o) to the 4β-aminopodophyllotoxin. The synthesized derivatives 13a-o were evaluated for their cytotoxicity against five human cancer cell lines (breast, oral, colon, lung and ovarian). These podophyllotoxin conjugates have shown promising activity with GI₅₀ values ranging from <0.1 to 0.29 μM. Some of the compounds 13j, 13k and 13l that showed significant antiproliferative activity were also evaluated for related cytotoxic effects in MCF-7 cells, and compared to etoposide. These compounds (13j, 13k and 13l) showed G2/M cell cycle arrest and the apoptotic event was found to be due to both the single-strand DNA breaks as observed by comet assay as well as double-strand breaks as observed by the large accumulation of gamma H2AX foci.

Carbazole–pyrrolo[2,1-c][1,4]benzodiazepine conjugates: design, synthesis, and biological evaluation

A series of carbazole–pyrrolobenzodiazepine conjugates (4a–g and 5a–f) have been designed, and synthesized as anticancer agents. These compounds are prepared by linking the C8-position of DC-81 with a carbazole moiety through simple alkane spacers as well as piperazine side-armed alkane spacers in good yields. The DNA binding ability of these conjugates has been determined by thermal denaturation studies and also supported by molecular docking studies. These conjugates showed potent anticancer activity with GI50 ranging from 5.27–0.01 μM. The FACS analysis and BrdU assay of selected conjugates (4c, 4f, 5a and 5f) on MCF-7 cell lines disclosed the increased G1 cell cycle arrest and one of the conjugates 5f has exhibited significant anticancer activity. The analysis of the intrinsic factors involved in causing the G1 arrest in MCF-7 cell lines by 5f conjugate has been demonstrated on the proteins which play a vital role in G1 arrest followed by apoptosis (Cyclin D1, CDK4, c-Jun, JunB, CREB, p53, JNK1/2, procaspase-7, cleaved PARP, pRb, and BAX). Thus, these PBD conjugates (in particular 5f) have promising potency for combating human carcinoma.

Quinazolino linked 4β-amidopodophyllotoxin conjugates regulate angiogenic pathway and control breast cancer cell proliferation.

A series of new conjugates of quinazolino linked 4β-amidopodophyllotoxins 10aa-af and 10ba-bf were synthesized and evaluated for their anticancer activity against human pancreatic carcinoma (Panc-1) as well as breast cancer cell lines such as MCF-7 and MDA-MB-231 by employing MTT assay. Among these conjugates, some of them like 10bc, 10bd, 10be and 10bf exhibited high potency of cytotoxicity. Flow cytometric analysis showed that these conjugates arrested the cell cycle in the G2/M phase and caused the increase in expression of p53 and cyclin B1 protein with concomitant decrease in Cdk1 thereby suggesting the inhibitory action of these conjugates on mitosis. Interestingly, we observed a decrease in expression of proteins that control the tumor micro environment such as VEGF-A, STAT-3, ERK1/2, ERK-p, AKT-1 ser 473 phosphorylation in compounds treated breast cancer cells. Further, these effective conjugates have exhibited inhibitory action on integrin (αVβIII). Furthermore, the MCF-7 cells that were arrested and lost the proliferative capacity undergo mitochondrial mediated apoptosis by activation of caspases-9. Thus these conjugates have the potential to control breast cancer cell growth by effecting tumor angiogenesis and invasion.

Chalcone-imidazolone conjugates induce apoptosis through DNA damage pathway by affecting telomeres.

BackgroundBreast cancer is one of the most prevalent cancers in the world and more than one million women are diagnosed leading to 410,000 deaths every year. In our previous studies new chalcone-imidazolone conjugates were prepared and evaluated for their anticancer activity in a panel of 53 human tumor cell lines and the lead compounds identified were 6 and 8. This prompted us to investigate the mechanism of apoptotic event.ResultsInvolvement of pro-apoptotic protein (Bax), active caspase-9 and cleavage of retinoblastoma protein was studied. Interestingly, the compounds caused upregulation of p21, check point proteins (Chk1, Chk2) and as well as their phosphorylated forms which are known to regulate the DNA damage pathway. Increased p53BP1 foci by immunolocalisation studies and TRF1 suggested the possible involvement of telomere and associated proteins in the apoptotic event. The telomeric protein such as TRF2 which is an important target for anticancer therapy against human breast cancer was extensively studied along with proteins involved in proper functioning of telomeres.ConclusionsThe apoptotic proteins such as Bax, active caspase-9 and cleaved RB are up-regulated in the compound treated cells revealing the apoptotic nature of the compounds. Down regulation of TRF2 and upregulation of the TRF1 as well as telomerase assay indicated the decrease in telomeric length revealing telomeric dysfunction and thereby controlling the rapid rate of cell proliferation. In summary, chalcone-imidazolone conjugates displayed significant DNA damage activity particularly at telomeres and caused both apoptosis and senescence-like growth arrest which suggested that these compounds have potential activity against breast carcinoma.

Synthesis, anticancer activity and mitochondrial mediated apoptosis inducing ability of 2,5-diaryloxadiazole–pyrrolobenzodiazepine conjugates

A series of 2,5-diaryloxadiazole linked pyrrolo[2,1-c][1,4]benzodiazepine conjugates have been prepared and evaluated for their anticancer activity. These conjugates have shown promising activity with GI50 values ranging from <0.1 to 0.29 microM. It is observed that some of these conjugates particularly 4a, 4d, 4i and 4l exhibit significant anticancer activity. Some detailed biological assays relating to the cell cycle aspects associated to Bax and caspases have been examined with a view to understand the mechanism of action of these conjugates.

Synthesis and biological evaluation of estradiol linked pyrrolo[2,1-c][1,4]benzodiazepine (PBD) conjugates as potential anticancer agents

A series of new estradiol linked pyrrolo[2,1-c][1,4]benzodiazepine (E(2)-PBD) conjugates (3a-f, 4a-f and 5a-f) with different linker architectures including a triazole moiety have been designed and synthesized. All the 18 compounds have been evaluated for their anticancer activity and it is observed that some of the compounds particularly 4c-e and 5c,d exhibited significant anticancer activity. The detailed biological aspects relating to the cell cycle effects and tubulin depolymerization activity have been examined with a view to understand the mechanism of action of these conjugates. Among all these conjugates, one of the compound 5c could be considered as the most effective compound particularly against MCF-7 breast cancer cell line.

Regulation of Cell Proliferation and Migration by miR-203 via GAS41/miR-10b Axis in Human Glioblastoma Cells.

Glioma amplified sequence 41(GAS41) is a potent transcription factor that play a crucial role in cell proliferation and survival. In glioblastoma, the expression of GAS41 at both transcriptional and post transcriptional level needs to be tightly maintained in response to cellular signals. Micro RNAs (miRNA) are small non coding RNA that act as important regulators for modulating the expression of various target genes. Studies have shown that several miRNAs play role in the post-transcriptional regulation of GAS41. Here we identified GAS41 as a novel target for endogenous miR-203 and demonstrate an inverse correlation of miR-203 expression with GAS41 in glioma cell lines (HNGC2 and U87). Over expression of miR-203 negatively regulates GAS41 expression in U87 and HNGC2 cell lines. Moreover, miR-203 restrained miR-10b action by suppressing GAS41. GAS41 is essential for repressing p53 in tumor suppressor pathway during cell proliferation. Enforced expression of GAS41 produced contradictory effect on miR-203 but was able to enhance p53 tumor suppressor pathway associated protein. It was also found that miR-203 maintains the stability of p53 as knock down of p53 expression using siRNA resulted in down regulation of pri-miR and mature miR-203 expression. Conversely reconstitution of miR-203 expression induced apoptosis and inhibited migratory property of glioma cells. Taken together, we show that miR-203 is a key negative regulator of GAS41 and acts as tumor suppressor microRNA in glioma.

Bisindole-PBD regulates breast cancer cell proliferation via SIRT-p53 axis

In a previous study we reported the role of potent bisindole-PBD conjugate as an inclusion in the arsenal of breast cancer therapeutics. In breast cancer cell proliferation, PI3K/AKT/mTOR pathway plays a crucial role by prosurvival mechanism that inhibits programmed cell death. Here, 2 breast cancer cells lines, MCF-7 and MDA-MB-231 were treated with Vorinostat (suberoylanilide hydroxamic acid / SAHA) and bisindole-PBD (5b). We have investigated the effect on PI3K/AKT/mTOR pathway and SIRT expression including epigenetic regulation. There was consistent decrease in the level of PI3K, AKT, mTOR proteins upon treatment of 5b in both MCF-7 and MDA-MB-231 cell lines compared to untreated controls. Treatment with caspase inhibitor (Q-VD-OPH) confirmed that the effect of 5b on PI3K signaling was ahead of apoptosis. Real time PCR and western blot analysis showed profound reduction in the mRNA and protein levels of SIRT1 and SIRT2. Molecular docking studies also supported the interaction of 5b with various amino acids of SIRT2 proteins. Treatment with 5b caused epigenetic changes that include increase of acetylated forms of p53, increase of histone acetylation at p21 promoter as well as decrease in methylation state of p21 gene. Compound 5b thus acts as SIRT inhibitor and cause p53 activation via inhibition of growth factor signaling and activation of p53 dependent apoptotic signaling. This present study focuses bisindole-PBD on epigenetic alteration putting 5b as a promising therapeutic tool in the realm of breast cancer research.

A novel bisindole-PBD conjugate inhibits angiogenesis by regulating STAT3 and VEGF in breast cancer cells

AIMS Breast cancer is highly resistant to chemotherapeutic approach and hence, alternative strategies have been developed to fight against this heterogeneous group of disease. In particular, many studies have demonstrated about various drugs for the treatment of breast cancer. In our study, we assessed the anti-angiogenenic activities of Bisindole-PBD (5b) in MCF-7 and MDA-MB-231 cell lines. MAIN METHODS In vitro Endothelial Cell (HUVEC) Tube Formation Assay was performed to show inhibitory role of 5b along with its role upon wound healing process in breast cancer cells in vitro. Semi-quantitative reverse transcription PCR (RT-PCR) was also done to examine the expression of VEGF in response to 5b in breast cancer cells and in HUVEC cells. siRNA transfection study explored STAT3 mediated VEGF transcription in breast cancer cells MCF-7 and MDA-MB-231. CAM assay was performed to see the role of 5b on vessel formation in chicken embryo. KEY FINDINGS From in vitro data we have demonstrated that 5b played a role in regulation of breast cancer cell proliferation by inhibiting angiogenesis. Test drug 5b suppressed the expression VEGF at both transcriptional and post transcriptional levels. Apart from this, there was significant down regulation in STAT3 level after drug treatment, which was found to be involved in the VEGF transcription. Metastasis related MMP-2 and MMP-9 expressions were also modulated by 5b. In vivo study by Chick Chorioallantoic Membrane (CAM) Assay also showed anti-angiogenesis role of the test drug which was consistent with the in vitro data. SIGNIFICANCE Altogether, our data demonstrated 5b as potent small molecule with anti-angiogenic activities.