This study investigates the efficacy of carnosic acid (CA), a polyphenolic diterpene, isolated from the plant rosemary (Rosemarinus officinalis), on androgen-independent human prostate cancer PC-3 cells. CA induced anti-proliferative effects in PC-3 cells in a concentration- and time-dependent manner, which was due to apoptotic induction as evident from flow-cytometry, DNA laddering and TUNEL assay. Apoptosis was associated with the activation of caspase-8, -9, -3 and -7, increase in Bax:Bcl-2 ratio, release of cytochrome-c and decrease in expression of inhibitor of apoptosis (IAP) family of proteins. Apoptosis was attenuated upon pretreatment with specific inhibitors of caspase-8 (Z-IETD-fmk) and caspase-9 (Z-LEHD-fmk) suggesting the involvement of both intrinsic and extrinsic apoptotic cascades. Further, apoptosis resulted from the inhibition of IKK/NF-κB pathway as evident from decreased DNA binding activity, nuclear translocation of p50 and p65 and IκBα phosphorylation. The down-regulation of IKK/NF-κB was associated with inhibition of Akt phosphorylation and its kinase activity with a concomitant increase in the serine/threonine protein phosphatase 2A (PP2A) activity. Pharmacologic inhibition of PP2A by okadaic acid and calyculin A, significantly reversed CA-mediated apoptotic events in PC-3 cells indicating that CA induced apoptosis by activation of PP2A through modulation of Akt/IKK/NF-κB pathway. In addition, CA induced apoptosis in another androgen refractory prostate cancer DU145 cells via intrinsic pathway as evidenced from the activation of caspase 3, cleavage of PARP, increase in Bax:Bcl-2 ratio and cytochrome-c release. Carnosic acid, therefore, may have the potential for use in the prevention and/or treatment of prostate cancer.

TLRs, which form an interface between mammalian host and microbe, play a key role in pathogen recognition and initiation of proinflammatory response thus stimulating antimicrobial activity and host survival. However, certain intracellular pathogens such as Leishmania can successfully manipulate the TLR signaling, thus hijacking the defensive strategies of the host. Despite the presence of lipophosphoglycan, a TLR2 ligand capable of eliciting host-defensive cytokine response, on the surface of Leishmania, the strategies adopted by the parasite to silence the TLR2-mediated proinflammatory response is not understood. In this study, we showed that Leishmania donovani modulates the TLR2-mediated pathway in macrophages through inhibition of the IKK–NF-κB cascade and suppression of IL-12 and TNF-α production. This may be due to impairment of the association of TRAF6 with the TAK–TAB complex, thus inhibiting the recruitment of TRAF6 in TLR2 signaling. L. donovani infection drastically reduced Lys 63-linked ubiquitination of TRAF6, and the deubiquitinating enzyme A20 was found to be significantly upregulated in infected macrophages. Small interfering RNA-mediated silencing of A20 restored the Lys 63-linked ubiquitination of TRAF6 as well as IL-12 and TNF-α levels with a concomitant decrease in IL-10 and TGF-β synthesis in infected macrophages. Knockdown of A20 led to lower parasite survival within macrophages. Moreover, in vivo silencing of A20 by short hairpin RNA in BALB/c mice led to increased NF-κB DNA binding and host-protective proinflammatory cytokine response resulting in effective parasite clearance. These results suggest that L. donovani might exploit host A20 to inhibit the TLR2-mediated proinflammatory gene expression, thus escaping the immune responses of the host.

The role of phosphatases in the impairment of MAPK signaling, which is directly responsible for Leishmania-induced macrophage dysfunction, is still poorly understood. Gene expression profiling revealed that Leishmania donovani infection markedly up-regulated the expression of three phosphatases: MKP1, MKP3, and PP2A. Inhibition of these phosphatases prior to infection points toward preferential induction of the Th2 response through deactivation of p38 by MKP1. On the other hand, MKP3 and PP2A might play significant roles in the inhibition of iNOS expression through deactivation of ERK1/2. Among various PKC isoforms, PKCzeta was associated with induction of MKP3 and PP2A in infected macrophages, whereas PKCepsilon was correlated with MKP1 induction. Inhibition of phosphatases in L. donovani-infected BALB/c mice shifted the cytokine balance in favor of the host by inducing TNF-alpha and iNOS expression. This was validated by cystatin, an immunomodulator and curing agent for experimental visceral leishmaniasis, which showed that inhibition of MKPs and PP2A activity may be necessary for a favorable T cell response and suppression of organ parasite burden. This study, for the first time, suggests the possibility of the involvement of MAPK-directed phosphatases in the establishment of L. donovani infection.

Triterpenes found in plants display a multitude of biological activities, including anti-tumor properties. The present study investigates the effect of 18β-glycyrrhetinic acid (GRA) a pentacyclic triterpenoid of the β-amyrin type, isolated from the root of Licorice (Glycyrrhizza glabra) on human breast cancer cells, MCF-7. GRA showed potent inhibitory effects on MCF-7 proliferation in a concentration- and time-dependent manner without affecting immortalized normal mammary epithelial cell line (MCF-10A). Growth inhibition of MCF-7 cells by GRA occurred through apoptosis, as evident from phosphatidyl serine externalization and DNA fragmentation. Apoptosis was primarily mediated through mitochondrial death cascade as evidenced by loss of mitochondrial membrane potential, release of cytochrome c and activation of caspase-9. GRA induced an increase in Bax:Bcl-2 ratio along with a significant increase in the protein level of the BH3 protein Bim. SiRNA-mediated knock down of Bim markedly attenuated GRA-mediated apoptosis. Profiling of transcriptional regulators of Bim revealed a role of Forkhead box O 3a transcription factor (FOXO3a) as judged by increased expression and nuclear translocation of FOXO3a. Silencing of FOXO3a resulted in marked attenuation in the expression of Bim as well as protection against GRA-mediated apoptosis. Furthermore, GRA-induced activation and nuclear localization of FOXO3a was associated with a reduced activity of Akt kinase. These results suggest that GRA induces apoptosis in human breast carcinoma MCF-7 cells via caspase activation and modulation of Akt/FOXO3a pathway.

Hesperetin, a flavanone glycoside predominantly found in citrus fruits, exhibits a wide array of biological properties. In the present study hesperetin exhibited a significant cytotoxic effect in human breast carcinoma MCF-7 cells in a concentration- and time-dependent manner without affecting normal (HMEC) as well as immortalized normal mammary epithelial cells (MCF-10A). The cytotoxic effect of hesperetin was due to the induction of apoptosis as evident from the phosphatidyl-serine externalization, DNA fragmentation, caspase-7 activation, and PARP cleavage. Apoptosis was associated with caspase-9 activation, mitochondrial membrane potential loss, release of cytochrome c, and increase in Bax:Bcl-2 ratio. Pre-treatment with caspase-9 specific inhibitor (Z-LEHD-fmk) markedly attenuated apoptosis suggesting an involvement of intrinsic mitochondrial apoptotic cascade. Further, DCFDA flow-cytometric analysis revealed triggering of ROS in a time-dependent manner. Pre-treatment with ROS scavenger N-acetylcysteine (NAC) and glutathione markedly abrogated hesperetin-mediated apoptosis whereas carbonyl cyanide m-chlorophenylhydrazone (CCCP) pretreatment along with DHR123-based flow-cytometry indicated the generation of cytosolic ROS. Profiling of MAPKs revealed activation of JNK upon hesperetin treatment which was abrogated upon NAC pre-treatment. Additionally, inhibition of JNK by SP600125 significantly reversed hesperetin-mediated apoptosis. The activation of JNK was associated with the activation of ASK1. Silencing of ASK1 resulted in significant attenuation of JNK activation as well as reversed the hesperetin-mediated apoptosis suggesting that hesperetin-mediated apoptosis of MCF-7 cells involves accumulation of ROS and activation of ASK1/JNK pathway. In addition, hesperetin also induced apoptosis in triple negative breast cancer MDA-MB-231 cells via intrinsic pathway via activation of caspase -9 and -3 and increase in Bax:Bcl-2 ratio.