Thiol antioxidants sensitize malabaricone C induced cancer cell death via reprogramming redox sensitive p53 and NF-κB proteins in vitro and in vivo.

Specific focus on "redox cancer therapy" by targeting drugs to redox homeostasis of the cancer cells is growing rapidly. Recent clinical studies showed that N-acetyl cysteine (NAC) treatment significantly decreased the metabolic heterogeneity and reduced Ki67 (a proliferation marker) with simultaneous enhancement in apoptosis of tumor cells in patients. However, it is not yet precisely known how thiol antioxidants enhance killing of cancer cells in a context dependent manner. To this end, we showed that a dietary compound, malabaricone C (mal C) generated copious amounts of reactive oxygen species (ROS) and also reduced GSH level in lung cancer cells. Paradoxically, although antioxidants supplementation reduced mal C-induced ROS, thiol-antioxidants (NAC/GSH) restored intracellular GSH level but enhanced DNA DSBs and apoptotic cell death induced by mal C. Our results unraveled two tightly coupled biochemical mechanisms attributing this sensitization process by thiol antioxidants. Firstly, thiol antioxidants enable the "catechol-quinone redox cycle" of mal C and ameliorate ROS generation and bio-molecular damage (DNA and protein). Secondly, thiol antioxidants cause rapid glutathionylation of transcription factors [p53, p65 (NF-κB) etc.], oxidized by mal C, and abrogates their nuclear sequestration and transcription of the anti-apoptotic genes. Furthermore, analyses of the mitochondrial fractions of p53 expressing and silenced cells revealed that cytoplasmic accumulation of glutathionylated p53 (p53-SSG) triggers a robust mitochondrial death process. Interestingly, mutation of redox sensitive cysteine residues at 124, 141 and 182 position in p53 significantly reduces mal C plus NAC mediated sensitization of cancer cells. The preclinical results, in two different tumor models in mice, provides further support our conclusion that NAC is able to sensitize mal C induced suppression of tumor growth in vivo.

Spice-derived phenolic, malabaricone B induces mitochondrial damage in lung cancer cells via a p53-independent pathway.

The spice-derived phenolic, malabaricone B (mal B) showed selective toxicity to human lung cancer (A549), malignant melanoma (A375) and T cell leukemia (Jurkat) cell lines, without showing toxicity to human normal intestinal (INT407), human kidney (HEK293) and lung fibroblast (WI-38) cells. Among the chosen cancer cell lines, mal B showed maximum cytotoxicity to the A549 cells (IC50 = 8.1 ± 1.0 µM), which was significantly better than that of curcumin (IC50 = 26.7 ± 3.1 µM). Further morphological studies by phase contrast microscopy and a clonogenic assay of the A549 cells revealed that mal B treatment increased the number of shrinking cells and also abolished the clonal proliferation of the cells. Mal B induced apoptotic cell death was confirmed by DNA laddering and quantified by cytoplasmic oligonucleosome formation and annexin V/PI assays. The mal B-induced apoptosis was mediated by an increase in the intracellular reactive oxygen species (ROS), because the cell-permeable antioxidants, N-acetylcysteine (NAC) and PEG-SOD, strongly inhibited its cytotoxicity to the A549 cells. Mal B increased the BAX level while simultaneously decreasing the BCL-2 and BCL-XL levels in the A549 cells, triggering the mitochondrial apoptotic pathway as revealed from the release of cytochrome c, and the activation of caspase-9 and caspase-3. Pre-treatment of cells with caspase-9, caspase-3 and pan-caspase inhibitors made them more resistant to mal B treatment. This effect of mal B was strongly associated with the concomitant decrease in anti-apoptotic (IAP1, IAP2 and survivin), angiogenic (growth factors) and cancer invasiveness (matrix metalloproteinase-9, COX-2) modulating proteins. Mal B induced cytotoxicity was unaffected by the shRNA-mediated depletion of p53 in A549 cells. Most importantly, mal B sensitized a wide range of human carcinoma cells regardless of their p53 status. Finally, mal B (100 mg kg-1) also inhibited lung tumor (xenograft) growth in SCID mice.

DNA damage dependent activation of checkpoint kinase-1 and mitogen-activated protein kinase-p38 are required in malabaricone C-induced mitochondrial cell death.

BACKGROUND: Given that lung cancer is the second leading cause of cancer-related deaths with low survival rates, the project was aimed to formulate an efficient drug with minimum side effects, and rationalize its action mechanistically. METHODS: Mitochondria deficient cells, shRNA-mediated BCL2 and ATM depleted cells and pharmacological inhibition of DNA-damage response proteins were employed to explore the signaling mechanism governed between nucleus and mitochondria in response to mal C. RESULTS: Mal C decreased cell viability in three lung carcinoma cells, associated with DNA damage, p38-MAPK activation, imbalance in BAX/BCL2 expression, mitochondrial dysfunction and cytochrome-c release. Mitochondria depletion and p38-MAPK inhibition made A549 cells extremely resistant, but BCL2 knock-down partially sensitized the cells to mal C treatment. The mal C-induced apoptosis in A549 cells was initiated by DNA single strand breaks that led to double strand breaks (DSBs). DSB generation paralleled the induction of ATM- and ATR-mediated CHK1 phosphorylation. ATM silencing and ATR inhibition partially attenuated the mal C-induced p38-MAPK activation, CHK1 phosphorylation and apoptosis, which were completely suppressed by CHK1 inhibition. CONCLUSIONS: Mal C activates the ATM-CHK1-p38 MAPK cascade to cause mitochondrial cell death in lung carcinoma cells. GENERAL SIGNIFICANCE: Given that mal C has appreciable natural abundance and is non-toxic to mice, further in vivo evaluation would help in establishing its anti-cancer property.

Antipromastigote activity of the malabaricones of Myristica malabarica (rampatri).

A major problem in the management of visceral leishmaniasis, especially in the Indian subcontinent, is the growing unresponsiveness to conventional antimonial therapy, indicating the urgent need to identify new antileishmanial compounds. This study was undertaken to evaluate the antileishmanial activity of the fruit rind of Myristica malabarica that is used as a spice and is also credited with medicinal properties. The antipromastigote activity of different extracts/fractions of M. malabarica and its constituent diarylnonanoids were evaluated in Leishmania donovani promastigotes (MHOM/IN/83/AG83) using the MTS-PMS assay. Preliminary screening of the ether extract (R1) with its crude methanol fraction (R2) and two fractions (R3 and R4) revealed that R2 had potent leishmanicidal activity (IC(50) 31.0 microg/mL), whereas R3 and R4 showed poor activity. Fractionation of R2 yielded four diarylnonanoids (malabaricones A-D, designated as Mal A, Mal B, Mal C and Mal D, respectively). The IC(50) values of Mal A-D were 16, 22, 27 and >50 microg/mL, respectively. Taken together, the data suggest that the methanol extract of M. malabarica, especially its constituent compounds, Mal A and Mal B, have promising antileishmanial activity meriting further investigations regarding the underlying molecular mechanism(s) of action with a view towards future drug development.