Summary Prostate apoptosis response-4 (Par-4) is a pro-apoptotic protein with intracellular functions in the cytoplasm and nucleus. Unexpectedly, we noted Par-4 protein is spontaneously secreted by normal and cancer cells in culture, and by Par-4 transgenic mice that are resistant to spontaneous tumors. Short exposure to endoplasmic reticulum (ER) stress-inducing agents further increased cellular secretion of Par-4 by a brefeldin A-sensitive pathway. Secretion occurred independently of caspase activation and apoptosis. Interestingly, extracellular Par-4 induced apoptosis by binding to the stress response protein, glucose-regulated protein-78 (GRP78), expressed at the surface of cancer cells. The interaction of extracellular Par-4 and cell surface GRP78 led to apoptosis via ER stress and activation of the FADD/caspase-8/caspase-3 pathway. Moreover, apoptosis inducible by TRAIL, which also exerts cancer cell-specific effects, is dependent on extracellular Par-4 signaling via cell surface GRP78. Thus, Par-4 activates a novel extrinsic pathway involving cell surface GRP78 receptor for induction of apoptosis.
Activation of the PI3K-Akt pathway by loss of tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) function, increased growth factor signaling, or oncogene expression renders cancer cells resistant to apoptotic signals and promotes tumor growth. Although Akt acts as a global survival signal, the molecular circuits of this pathway have not been completely established. We report that Akt physically binds to the pro-apoptotic protein Par-4 via the Par-4 leucine zipper domain and phosphorylates Par-4 to inhibit apoptosis. Suppression of Akt activation by the PI3K-inhibitor PTEN or LY294002, Akt expression by RNA-interference, or Akt function by dominant-negative Akt caused apoptosis in cancer cells. Apoptosis induced by inhibiting Akt was blocked by inhibition of Par-4 expression, but not by inhibition of other apoptosis agonists that are Akt substrates, suggesting that inhibition of the PI3K-Akt pathway leads to Par-4-dependent apoptosis. Thus, Par-4 is essential for PTEN-inducible apoptosis, and inactivation of Par-4 by Akt promotes cancer cell survival.
Tumors often show heterogeneity at many levels, including morphology, expression of prosurvival traits, and down-modulation of apoptotic or tumor suppressor traits. Despite tumor heterogeneity, however, several predominant traits exist within similar types of cancers. Such traits are prospective targets for therapeutic intervention strategies. However, because most cancer therapy protocols have an accompanying toxicity for normal cells, there are ongoing efforts directed toward the identification of apoptotic molecules that are highly selective for cancer cells. Our recent studies have identified Par-4 protein as a unique proapoptotic molecule that induces apoptosis in hormone-independent but not hormone-dependent cancer cells or in primary or immortalized normal cells (12). We describe here a novel mechanism underlying the selective activation of the apoptotic function of Par-4 in cancer cells.
Luteolin, a naturally occurring flavonoid, is abundant in our daily dietary intake. It exhibits a wide spectrum of pharmacological properties, but little is known about its biochemical targets other than the fact that it induces topoisomerase II-mediated apoptosis. In the present study, we show that luteolin completely inhibits the catalytic activity of eukaryotic DNA topoisomerase I at a concentration of 40 microM, with an IC50 of 5 microM. Preincubation of enzyme with luteolin before adding a DNA substrate increases the inhibition of the catalytic activity (IC50=0.66 microM). Treatment of DNA with luteolin before addition of topoisomerase I reduces this inhibitory effect. Subsequent fluorescence tests show that luteolin not only interacts directly with the enzyme but also with the substrate DNA, and intercalates at a very high concentration (>250 microM) without binding to the minor groove. Direct interaction between luteolin and DNA does not affect the assembly of the enzyme-DNA complex, as evident from the electrophoretic mobility-shift assays. Here we show that the inhibition of topoisomerase I by luteolin is due to the stabilization of topoisomerase-I DNA-cleavable complexes. Hence, luteolin is similar to camptothecin, a class I inhibitor, with respect to its ability to form the topoisomerase I-mediated 'cleavable complex'. But, unlike camptothecin, luteolin interacts with both free enzyme and substrate DNA. The inhibitory effect of luteolin is translated into concanavalin A-stimulated mouse splenocytes, with the compound inducing SDS-K+-precipitable DNA-topoisomerase complexes. This is the first report on luteolin as an inhibitor of the catalytic activity of topoisomerase I, and our results further support its therapeutic potential as a lead anti-cancer compound that poisons topoisomerases.
BackgroundWithaferin A, which is a naturally derived steroidal lactone, has been found to prevent angiogenesis and metastasis in diverse tumor models. It has also been recognized by different groups for prominent anti-carcinogenic roles. However, in spite of these studies on withanolides, their detailed anti-metastatic mechanism of action remained unknown. The current study has poised to address the machinery involved in invasion regulation by stable derivative of Withaferin A, 3-azido Withaferin A (3-azidoWA) in human cervical HeLa and prostate PC-3 cells.Methods and Principal FindingsSub-toxic concentration of 3-azidowithaferin A (3-azido WA) inhibited cancer cell motility and invasion in wound healing and Boyden chamber invasion by suppressing MMP-2 activity in gelatin zymography and its expression has proved to be a major obstacle in chemo-sensitivity. We have uncovered a novel mechanism of 3-azidoWA induced extracellular pro-apoptotic candidate tumor suppressor Par-4 protein stimulation in conditioned media and also noticed a concomitant marked reduction in pAkt and pERK signaling by immunoblot analysis. Furthermore, our zymography results suggest 3-azidoWA induced MMP-2 inhibition was mediated through secretory Par-4. The inhibition of apoptosis by 3-azidoWA could not restore MMP-2 gelatinase activity. In addition to this, our in vivo animal experiments data showed 3-azidoWA abrogated neovascularisation in dose dependent manner in mouse Matrigel plug assay.Conclusion/SignificanceFor this report, we found that 3-azidoWA suppressed motility and invasion of HeLa and PC-3 cells in MMP-2 dependent manner. Our in vitro result strongly suggests that sub-toxic doses of 3-azidoWA enhanced the secretion of extracellular Par-4 that abolished secretory MMP-2 expression and activity. Depletion of secretory Par-4 restored MMP-2 expression and invasion capability of HeLa and PC-3 cells. Further, our findings implied that 3-azidoWA attenuated internal phospho-ERK and phospho-Akt expression in a dose dependent manner might play a key role in inhibition of mouse angiogenesis by 3-azidoWA.
Ras is one of the most commonly mutated oncogenes in the array of human cancers. The mechanism by which Ras induces cellular transformation is, however, not fully elucidated. We present here evidence that oncogenic Ras suppresses the expression of the tumor suppressor phosphatase and tensin homologue deleted from chromosome 10
Leishmaniasis is the second-most dreaded parasitic disease in the modern world, behind malaria. The lack of effective vaccines demand improved chemotherapy along with the development of lead compounds and newer targets. We report here that the pentacyclic triterpenoid, dihydrobetulinic acid (DHBA), is a novel lead compound for antileishmanial therapy. It acts by targeting DNA topoisomerases. DNA topoisomerase I and II activity was studied using relaxation and decatenation assays. Mechanistic studies were based on the decreased mobility of enzyme-bound DNA compared with free DNA and the differential mobility of nicked and supercoiled monomers in 1% agarose gel. Pulsed field gradient gel electrophoresis, confocal microscopy, and transmission electron microscopy were performed to assess cytotoxicity of the compound and ultrastructural damage of the parasite. Apoptosis was studied by the isolation of DNA from DHBA-treated parasites and subsequent electrophoresis in 1% agarose gel. DHBA inhibits growth of Leishmania donovani promastigotes and amastigotes with an IC 50 of 2.6 and 4.1 M respectively. The compound is a dual inhibitor of DNA topoisomerases that fails to induce DNA cleavage and acts by preventing the formation of enzyme-DNA binary complex, ultimately inducing apoptosis. Treatment of infected golden hamsters with the compound markedly reduces (> 92%) parasitic burden, both in spleen and liver. Interestingly, the 17-decarboxylated analogue, dihydrolupeol, does not inhibit DNA topoisomerase I and II, has no effect on parasitic growth, and also fails to induce apoptosis. DHBA is a potent antileishmanial agent that induces apoptosis by primarily targeting DNA topoisomerases. Therefore it is a strong candidate for use in designing new antileishmanial drugs.
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