Susmita Mondal

Natural Products: Promising Resources for Cancer Drug Discovery

Natural products are important sources of anti-cancer lead molecules. Many successful anti-cancer drugs are natural products or their analogues. Many more are under clinical trials. The present review focuses on chemopreventive and anti-cancer activities of polar and non-polar extracts, semi purified fractions and pure molecules from terrestrial plants of India reported between 2005 and 2010 emphasizing possible mechanisms of action of pure molecules.

Withanolide D induces apoptosis in leukemia by targeting the activation of neutral sphingomyelinase-ceramide cascade mediated by synergistic activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase

BackgroundCeramide is an important second messenger that has diverse cellular and biological effect. It is a specific and potent inducer of apoptosis and suppressor of cell growth. In leukemia, chemoresistance generally developed due to deregulated ceramide metabolism. In combinatorial treatment strategies of leukemia, few components have the capability to increases ceramide production. Manipulation in ceramide production by physiological and pharmacological modulators therefore will give additive effect in leukemia chemotherapy.ResultsHere, we show that Withanolide D (C4β-C5β,C6β-epoxy-1-oxo-,20β, dihydroxy-20S,22R-witha-2,24-dienolide; WithaD), a pure herbal compound isolated from Withania somnifera could effectively induces apoptosis in a dose and time dependant manner both in myeloid (K562) and lymphoid (MOLT-4) cells being nontoxic to normal lymphocytes and control proliferative cells. WithaD potentially augment ceramide production in these cells. Downstream of ceramide, WithaD acted on MKK group of proteins and significantly increased JNK and p38MAPK phosphorylation. Pharmacological inhibition of p38MAPK and JNK proves their cooperative action on WithaD-induced cell death. Dissecting the cause of ceramide production, we found activation of neutral sphingomyelinase and showed neutral-sphingomyelinase 2 (N-SMase 2) is a critical mediator of WithaD-induced apoptosis. Knockdown of N-SMase 2 by siRNA and inhibitor of N-SMase (GW4869) significantly reduced WithaD-induced ceramide generation and phosphorylation of MKK4 and MKK3/6, whereas phosphorylation of MKK7 was moderately regulated in leukemic cells. Also, both by silencing of N-SMase 2 and/or blocking by GW4869 protects these cells from WithaD-mediated death and suppressed apoptosis, whereas Fumonisin B1, an inhibitor of ceramide synthase, did not have any effect. Additionally, WithaD effectively induced apoptosis in freshly isolated lymphoblasts from patients and the potent cell killing activity was through JNK and p38MAPK activation.ConclusionOur results demonstrate that WithaD enhance the ceramide accumulation by activating N-SMase 2, modulate phosphorylation of the JNK and p38MAPK and induced apoptosis in both myeloid and lymphoid cells along with primary cells derived from leukemia patients. Taken together, this pure herbal compound (WithaD) may consider as a potential alternative tool with additive effects in conjunction with traditional chemotherapeutic treatment, thereby accelerate the process of conventional drug development.

Down regulation of membrane-bound Neu3 constitutes a new potential marker for childhood acute lymphoblastic leukemia and induces apoptosis suppression of neoplastic cells

Membrane‐linked sialidase Neu3 is a key enzyme for the extralysosomal catabolism of gangliosides. In this respect, it regulates pivotal cell surface events, including trans‐membrane signaling, and plays an essential role in carcinogenesis. In this report, we demonstrated that acute lymphoblastic leukemia (ALL), lymphoblasts (primary cells from patients and cell lines) are characterized by a marked down‐regulation of Neu3 in terms of both gene expression (−30 to 40%) and enzymatic activity toward ganglioside GD1a (−25.6 to 30.6%), when compared with cells from healthy controls. Induced overexpression of Neu3 in the ALL‐cell line, MOLT‐4, led to a significant increase of ceramide (+66%) and to a parallel decrease of lactosylceramide (−55%). These events strongly guided lymphoblasts to apoptosis, as we assessed by the decrease in Bcl2/Bax ratio, the accumulation of Neu3 transfected cells in the sub G0–G1 phase of the cell cycle, the enhanced annexin‐V positivity, the higher cleavage of procaspase‐3. Therefore, the reduced expression of Neu3 in ALL could help lymphoblasts to survive, maintaining the cellular content of ceramide below a critical level. Interestingly, we found that Neu3 activity varied in relation to disease progression, increasing in clinical remission after chemotherapy, and decreasing again in patients that relapsed. In addition, a negative correlation was observed between Neu3 expression and the percentage of the ALL marker 9‐OAcGD3 positive cells. Consequently, Neu3 could represent a new potent biomarker in childhood ALL, to assess the efficacy of therapeutic protocols and to rapidly identify an eventual relapse.

Bak Compensated for Bax in p53-null Cells to Release Cytochrome c for the Initiation of Mitochondrial Signaling during Withanolide D-Induced Apoptosis

The goal of cancer chemotherapy to induce multi-directional apoptosis as targeting a single pathway is unable to decrease all the downstream effect arises from crosstalk. Present study reports that Withanolide D (WithaD), a steroidal lactone isolated from Withania somnifera, induced cellular apoptosis in which mitochondria and p53 were intricately involved. In MOLT-3 and HCT116p53+/+ cells, WithaD induced crosstalk between intrinsic and extrinsic signaling through Bid, whereas in K562 and HCT116p53−/− cells, only intrinsic pathway was activated where Bid remain unaltered. WithaD showed pronounced activation of p53 in cancer cells. Moreover, lowered apoptogenic effect of HCT116p53−/− over HCT116p53+/+ established a strong correlation between WithaD-mediated apoptosis and p53. WithaD induced Bax and Bak upregulation in HCT116p53+/+, whereas increase only Bak expression in HCT116p53−/− cells, which was coordinated with augmented p53 expression. p53 inhibition substantially reduced Bax level and failed to inhibit Bak upregulation in HCT116p53+/+ cells confirming p53-dependent Bax and p53-independent Bak activation. Additionally, in HCT116p53+/+ cells, combined loss of Bax and Bak (HCT116Bax−Bak−) reduced WithaD-induced apoptosis and completely blocked cytochrome c release whereas single loss of Bax or Bak (HCT116Bax−Bak+/HCT116Bax+Bak−) was only marginally effective after WithaD treatment. In HCT116p53−/− cells, though Bax translocation to mitochondria was abrogated, Bak oligomerization helped the cells to release cytochrome c even before the disruption of mitochondrial membrane potential. WithaD also showed in vitro growth-inhibitory activity against an array of p53 wild type and null cancer cells and K562 xenograft in vivo. Taken together, WithaD elicited apoptosis in malignant cells through Bax/Bak dependent pathway in p53-wild type cells, whereas Bak compensated against loss of Bax in p53-null cells.

Withanolide D, Carrying the Baton of Indian Rasayana Herb as a Lead Candidate of Antileukemic Agent in Modern Medicine

During cancer, immune system gets suppressed due to developing malignancy and anticancer drugs used. Therefore, an ideal chemotherapeutic agent would have both immuno-stimulatory and cancer cell death-inducing activity. Here, we have shown that Withanolide D (WithaD), an important bioactive withanolide, purified from the leaves of an Indian chemotype (NMITLI 135) of Withania somnifera acts as stimulator of Th1 response along with moderate effect on Th2 response, thus advocates its candidature as a potent immunostimulator. Additionally, WithaD exhibited antileukemic activity, targeting multiple pathways along with ceramide accumulation through N-SMase 2 activation, ultimately inducing apoptosis in neoplastic cells. Taken together, WithaD is a potential herbal compound with dual functions: potentiation of the intrinsic defense mechanism and inducer of cancer cell apoptosis.

mTORC2 regulates hedgehog pathway activity by promoting stability to Gli2 protein and its nuclear translocation

mTORC2 is aberrantly activated in cancer and therefore is considered to be an important therapeutic target. The hedgehog pathway, which is also often hyperactivated, regulates transcription of several genes associated with angiogenesis, metastasis, cellular proliferation and cancer stem cell (CSC) regeneration. However, the contribution of mTORC2 toward hedgehog pathway activity has not been explored yet. Here we have addressed the molecular cross talk between mTORC2 and hedgehog pathway activities in the context of glioblastoma multiforme, a malignant brain tumor using as a model system. We observed that higher mTORC2 activity enhanced the expression of a few hedgehog pathway molecules (Gli1, Gli2 and Ptch1) and amplified its target genes (Cyclin D1, Cyclin D2, Cyclin E, Snail, Slug and VEGF) both in mRNA and protein levels as corroborated by increased metastasis, angiogenesis, cellular proliferation and stem cell regeneration. Inhibition of mTORC2 formation decreased hedgehog pathway activity and attenuated all these above-mentioned events, suggesting their cross talk with each other. Further investigations revealed that mTORC2 inhibited ubiquitination of Gli2 by inactivating GSK3β, and thus it promotes stability to Gli2 and its nuclear translocation. Moreover, enhanced mTORC2 activity led to the increased clonogenic properties and CD133+ cells, indicating its role in CSC regeneration. mTORC2 inhibitor directed the reduction of hedgehog pathway proteins and also reduced CSCs. Thus, our observations support a role for elevated mTORC2 activity in regulating angiogenesis, metastasis, cellular proliferation and CSC regeneration via hedgehog pathway activity. Taken together, it provides a rationale for including the mTOR2 inhibitor as part of the therapeutic regimen for CSCs.

9- O -Acetyl GD3 in Lymphoid and Erythroid Cells

Sialic acids are electronegatively charged sugars that contribute to the enormous structural diversity of complex carbohydrates, which are major constituents of mostly proteins and lipids of cell membranes and secreted macromolecules. They are usually positioned at the outer end of these molecules and thus are well suited for interacting with other cells, pathogens, or molecules in the cell environment. Sialic acids are 9-carbon-containing monosaccharides, and the structural diversity of glycan chains is further increased by the various modifications of sialic acids [1]. Amongst 50 known derivatives of sialic acids, 7-, 8-, and 9-O-acetylated derivatives (O-AcSA) are important constituents of the cell membrane and are known to influence many physiological and pathological processes [1, 2], including cell–cell adhesion, signaling, differentiation, and metastasis [3–6]. However, as O-acetyl esters from positions C-7 and C-8 spontaneously migrate to C-9, even under physiologic conditions, O-acetylation at C-9 is considered the most common biologically occurring modification [7]. The appearance of O-acetylated sialic acids on glycoproteins or glycolipids is cell-type specific and developmentally regulated, their synthesis and turnover being a highly orchestrated phenomenon. O-acetylation can have a significant role in cell physiology and can alter the functional effects of important molecular determinants in various disease conditions. In this chapter, we deal with the O-acetylation of glycosphingolipids (GSLs), specifically GD3 in both erythroid and lymphoid cells.