Suman Kumar Samanta

Apoptotic effects of mahanine on human leukemic cells are mediated through crosstalk between Apo-1/Fas signaling and the Bid protein and via mitochondrial pathways

Apo-1 (Fas/CD95), a cell surface receptor, triggers apoptosis after binding to its physiological ligand, Apo-1L (FasL/CD95L). This study reports that mahanine, purified from the leaves of Murraya koenigii, has a dose- and time-dependent anti-proliferative activity in acute lymphoid (MOLT-3) and chronic myeloid (K562) leukemic cell lines and in the primary cells of leukemic and myeloid patients, with minimal effect on normal immune cells including CD34(+) cells. Leukemic cells underwent phosphatidylserine externalization and DNA fragmentation, indicating mahanine-induced apoptosis. An increase in reactive oxygen species suggests that the mahanine-induced apoptosis was mediated by oxidative stress. A significant drop in the Bcl2/Bax ratio, the loss of mitochondrial transmembrane potential as well as cytochrome c release from the mitochondria to the cytosol suggested involvement of the mitochondrial pathway of apoptosis. Cytochrome c release was followed by the activation of caspase-9, caspase-3 and caspase-7, and cleavage of PARP in both MOLT-3 and K562 cells. In MOLT-3 cells, formation of the Fas-FasL-FADD-caspase-8 heterotetramer occurred, leading to the cleavage of Bid to its truncated form, which consequently resulted in formation of the mitochondrial transmembrane pore. The incubation of MOLT-3 cells with mahanine in the presence of caspase-8 inhibitor or FasL-neutralizing NOK-2 antibody resulted in the decrease of mahanine-induced cell death. Mahanine was also a potent inhibitor of K562 xenograft growth, which was evident in an athymic nude mice model. In summary, these results provide evidence for involvement of the death receptor-mediated extrinsic pathway of apoptosis in the mahanine-induced anticancer activity in MOLT-3 cells, but not in K562 cells, which are deficient in Fas/FasL.

Oxidative inhibition of Hsp90 disrupts the super-chaperone complex and attenuates pancreatic adenocarcinoma in vitro and in vivo.

Pancreatic cancer is almost always fatal, in part because of its delayed diagnosis, poor prognosis, rapid progression and chemoresistance. Oncogenic proteins are stabilized by the Hsp90, making it a potential therapeutic target. We investigated the oxidative stress‐mediated dysfunction of Hsp90 and the hindrance of its chaperonic activity by a carbazole alkaloid, mahanine, as a strategic therapeutic in pancreatic cancer. Mahanine exhibited antiproliferative activity against several pancreatic cancer cell lines through apoptosis. It induced early accumulation of reactive oxygen species (ROS) leading to thiol oxidation, aggregation and dysfunction of Hsp90 in MIAPaCa‐2. N‐acetyl‐L‐cysteine prevented mahanine‐induced ROS accumulation, aggregation of Hsp90, degradation of client proteins and cell death. Mahanine disrupted Hsp90‐Cdc37 complex in MIAPaCa‐2 as a consequence of ROS generation. Client proteins were restored by MG132, suggesting a possible role of ubiquitinylated protein degradation pathway. Surface plasmon resonance study demonstrated that the rate of interaction of mahanine with recombinant Hsp90 is in the range of seconds. Molecular dynamics simulation showed its weak interactions with Hsp90. However, no disruption of the Hsp90‐Cdc37 complex was observed at an early time point, thus ruling out that mahanine directly disrupts the complex. It did not impede the ATP binding pocket of Hsp90. Mahanine also reduced in vitro migration and tube formation in cancer cells. Further, it inhibited orthotopic pancreatic tumor growth in nude mice. Taken together, these results provide evidence for mahanine‐induced ROS‐mediated destabilization of Hsp90 chaperone activity resulting in Hsp90‐Cdc37 disruption leading to apoptosis, suggesting its potential as a specific target in pancreatic cancer.

Mahanine, A DNA Minor Groove Binding Agent Exerts Cellular Cytotoxicity with Involvement of C-7-OH and −NH Functional Groups

Mahanine, a carbazole alkaloid is a potent anticancer molecule. To recognize the structure-activity correlation, mahanine was chemically modified. Antiproliferative activity of these derivatives was determined in 19 cancer cell lines from 7 different origins. Mahanine showed enhanced apoptosis compared to dehydroxy-mahanine-treated cells, indicating significant contribution of the C-7-OH group. O-Methylated-mahanine and N-methylated dehydroxy-mahanine-treated cells exhibited apoptosis only at higher concentrations, suggesting additional contribution of 9-NH group. Using biophysical techniques, we demonstrated that mahanine interacts with DNA through strong association with phosphate backbone compared to other derivatives but is unable to induce any conformational change in DNA, hence suggesting the possibility of being a minor groove binder. This was corroborated by molecular modeling and isothermal titration calorimetry studies. Taken together, the results of the current study represent the first evidence of involvement of C-7-OH and 9-NH group of mahanine for its cytotoxicity and its minor groove binding ability with DNA.

Improved chemosensitivity in cervical cancer to cisplatin: Synergistic activity of mahanine through STAT3 inhibition

Toxicity reduction of cisplatin is necessary for improved treatment of cancer. Here we have demonstrated the synergistic growth-inhibitory effect of cisplatin on cervical cancer cells in-combination with a nontoxic herbal carbazole alkaloid, mahanine. Mahanine enhanced cisplatin-induced apoptosis and reduced its effective concentration ∼5-8 folds. Mahanine inhibited JAK1 and Src and subsequently promoted proteasome-mediated degradation of STAT3. This event was further enhanced in-combination with cisplatin and subsequently inhibited cancer cell migration. Collectively, our results revealed that mahanine may be a prospective agent to reduce the concentration of cisplatin in adjunct for the treatment of cancer and thereby decreasing its toxicity.

Mahanine synergistically enhances cytotoxicity of 5-fluorouracil through ROS-mediated activation of PTEN and p53/p73 in colon carcinoma

Abstract5-Fluorouracil (5-FU) alone or in combination with other drugs is the main basis of chemotherapeutic treatment in colorectal cancer although patients with microsatellite instability generally show resistance to 5-FU treatment. The present investigation is focussed on the mechanistic insight of a pure herbal carbazole alkaloid, mahanine, as a single or in combination with 5-FU in colon cancer. We demonstrated that mahanine-induced apoptosis involved reactive oxygen species (ROS)-mediated nuclear accumulation of PTEN and its interaction with p53/p73. Mahanine and 5-FU in combination exerted synergistic inhibitory effect on cell viability. This combination also enhanced ROS production, increased tumour suppressor proteins and suppressed chemo-migration. Taken together, our results revealed that mahanine can be a potential chemotherapeutic agent with efficacy to reduce the concentration of toxic 5-FU in colon cancer.

Corchorusin-D, a saikosaponin-like compound isolated from Corchorus acutangulus Lam., targets mitochondrial apoptotic pathways in leukemic cell lines (HL-60 and U937)

PurposeThe presence of triterpene saponins in Corchorus acutangulus Lam. has been reported. However, no studies concerning biological activity of the plant extracts have been done so far. In the present study, the anti-leukemic activity of the methanol extract of aerial parts (ME) of C. acutangulus has been investigated, and efforts have been made to identify the active ingredient responsible for this activity.MethodsThe anti-leukemic activity of ME, its fractions and corchorusin-D (COR-D), the active ingredient, was investigated in leukemic cell lines U937 and HL-60 using cell viability and MTT assays. The molecular pathways leading to the activity of COR-D were examined by confocal microscopy, flow-cytometry, caspase and Western blot assays.ResultsME, its n-butanolic fraction and COR-D inhibited cell growth and produced significant cytotoxicity in leukemic cell lines U937 and HL-60. COR-D produced apoptotic cell death via mitochondrial disfunction and was found to pursue the intrinsic pathway by inciting the release of apoptosis-inducing factors (AIFs) from mitochondria. COR-D-induced translocation of Bax from cytosol to mitochondria facilitating caspase-9 activation and up regulation of downstream pathways leading to caspase-3 activation and PARP cleavage, which resulted in the subsequent accumulation of cells in the sub-G0 phase followed by DNA fragmentation.ConclusionsCOR-D possesses significant anti-leukemic activity in U937 and HL-60 cell lines by acting on the mitochondrial apoptotic pathways. Since the necrotic body formation is low after COR-D treatment, the occurrence of inflammation in in vivo systems could be reduced, which represents a positive indication in view of therapeutic application.

Identification and quantification of the active component quercetin 3-O-rutinoside from Barringtonia racemosa, targets mitochondrial apoptotic pathway in acute lymphoblastic leukemia

Barringtonia racemosa has been used as a traditional medicine for the treatment of various diseases. The antitumor property of the seed extract of this plant in mice model promotes us to search for the active component present in the fruit extract. Quercetin 3-O-rutinoside (QOR) has been isolated from the fruits of this plant for the first time and quantified by HPLC method. The compound was identified by IR, mass, and NMR (1D, 2D) spectral data analysis. QOR showed dose- and time-dependent anti-proliferative activity in several leukemic cell lines with negligible effect on normal human peripheral blood mononuclear cell (PBMC). A representative T-lineage acute lymphoblastic leukemia cell line (MOLT-3) showed phosphatidyl serine externalization and DNA fragmentation, indicating QOR-induced programed cell death. We established that QOR-induced apoptosis occurred preferentially on accumulation of cells in the sub-G0 phase and genomic DNA fragmentation through the activation of mitochondria-dependent caspase cascade for the first time in T-lineage ALL cell line.

Phytochemical Portfolio and Anticancer Activity of Murraya koenigii and its Primary Active Component, Mahanine

Graphical abstract Figure. No caption available. &NA; Murraya koenigii, a plant belonging to the Rutaceae family is widely distributed in Eastern‐Asia and its medicinal properties are well documented in Ayurveda, the traditional Indian system of medicine. Through systematic research and pharmacological evaluation of different parts of the plant extracts has been shown to possess antiviral, anti‐inflammatory, antioxidant, antidiabetic, antidiarrhoeal, antileishmanial, and antitumor activity. In the plant extracts, carbazole alkaloid, mahanine has been identified as the principle bioactive component among several other chemical constituents. Scientific evidence derived not only from in vitro cellular experiments but also from in vivo studies in various cancer models is accumulating for the pronounced anticancer effects of mahanine. The primary objective of this review is to summarize research data on cytotoxic chemical constituents present in different parts of Murraya koenigii and the anticancer activity of mahanine along with the recent understanding on the mechanism of its action in diverse cancer models. The information on its bioavailability and the toxicity generated from the recent studies have also been incorporated in the review.

Mahanine drives pancreatic adenocarcinoma cells into endoplasmic reticular stress-mediated apoptosis through modulating sialylation process and Ca 2+ -signaling

Endoplasmic reticulum (ER) stress results from protein unfolding/misfolding during cellular maturation, which requires a coordinated action of several chaperones and enzymes and Ca2+ signalling. ER-stress possibly has a positive effect on survival of pancreatic cancer cell. Therefore, detailed insights into this complex signaling network are urgently needed. Here, we systematically analyzed the impact of ER stress-mediated unfolded protein response (UPR) and Ca2+-signaling cross-talk for the survival of pancreatic adenocarcinoma (PDAC) cells. We observed enhanced ER activity and initiation of UPR signaling induced by a carbazole alkaloid (mahanine). This event triggers a time-dependent increase of intracellular Ca2+ leakage from ER and subsequently Ca2+ signaling induced by enhanced reactive oxygen species (ROS) produced by this pro-oxidant agent. In addition, we observed an altered glycosylation, in particular with regard to reduced linkage-specific sialic acids possibly due to decreased sialyltransferase activity. Changes in sialylation entailed enhanced expression of the ganglioside GD3 in the treated cells. GD3, an inducer of apoptosis, inhibited pancreatic xenograft tumor. Taken together, our study describes a molecular scenario how PDAC cells are driven into apoptosis by mahanine by UPR-driven ER stress-associated and ROS-mediated calcium signaling and possibly defective sialylation.