Amere Subbarao Sreedhar

Hsp90 isoforms: functions, expression and clinical importance

The 90 kDa heat shock protein, Hsp90, is a main functional component of an important cytoplasmic chaperone complex, and it is involved in various cellular processes, such as cell proliferation, differentiation and apoptosis. Identification of Hsp90 as a molecular target of various anticancer drugs highlighted its importance from the clinical point of view. Here we summarize the current knowledge on various Hsp90 isoforms regarding their genomic location, molecular evolution, functional differences, differential induction after various environmental stresses and in pathological conditions as well as the growing importance of discriminating between Hsp90 isoforms in clinical practice.

Apoptosis, necrosis and cellular senescence: chaperone occupancy as a potential switch

Chaperone function plays a key role in repairing proteotoxic damage and in the maintenance of cell survival. Here we compare the regulatory role of molecular chaperones (heat shock proteins, stress proteins) in cellular senescence, apoptosis and necrosis. We also review the current data on chaperone level and function in aging cells, and list some possible therapeutic interventions. Finally, we postulate a hypothesis, that increasing chaperone occupancy might be an important event which forces cells out of the normal cell cycle towards senescence. In the case of severe stress, this may lead to apoptosis or, following lethal stress, to cell necrosis.

Hsp90 inhibitors, GA and 17AAG, lead to ER stress-induced apoptosis in rat histiocytoma

Heat shock protein 90 (Hsp90) is a major molecular chaperone that plays an essential role in the maintenance of several signaling molecules, most of which are oncogenic kinases. Hsp90 inhibition by specific inhibitors leads to destabilization and loss of activity of such proteins, thereby leading to inhibition of multiple signaling cascades. Due to this, Hsp90 has emerged as an important target for the treatment of cancer. Inhibition of Hsp90 has been reported to induce apoptosis in certain cancer cell types. However, the molecular details of induction of apoptosis upon Hsp90 inhibition are not understood. We have investigated the effect of Hsp90 inhibition on a non-adherent rat histiocytoma cell line, BC-8, using geldanamycin and 17-Allylamino-17-demethoxygeldanamycin. We show that Hsp90 inhibition induces ER stress, which leads to disruption of mitochondrial homeostasis, leading to apoptosis. Induction of ER stress leads to increased expression of ER chaperones, Grp78 and Grp94, cleavage of caspase-12 and increase in cytoplasmic calcium. We show that calcium and Bax are responsible for the decrease in mitochondrial membrane potential (Deltapsi(m)), thereby leading to the release of cytochrome c and activation of caspase-9. Moreover, calcium chelator and over-expression of Bcl-2 is able to confer protection against apoptosis upon Hsp90 inhibition. We conclude that inhibition of Hsp90 leads to ER stress-induced mitochondria-mediated apoptosis and that Bax and Ca(2+) play an important role in mitochondrial damage.

Anti–microbial principles of selected remedial plants from Southern India

OBJECTIVE To examine the anti-bacterial activity of leaf extracts of Morus alba L. (Moraceae) and Piper betel L. (Piperaceae), and seed extracts of Bombax ceiba L. (Borabacaceae). METHODS We have partially purified plant extracts by solvent extraction method, and evaluated the effect of individual fractions on bacterial growth using Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) bacterial strains. RESULTS Compared with Morus and Bombax fractions, Piper fractions showed significant growth inhibition on all the three types of bacteria studied. The EtOAc-hexane fractions of Piper leaves exhibited significant anti-bacterial activity with minimum inhibitory concentrations (MIC) of 50 µg/mL culture against both gram-positive and gram-negative bacteria. The EtOAc-fractions I, II, and IV inhibited bacterial colony formation on soft agar in addition to growth inhibition. A combination treatment of piper fractions with ampicillin resulted in significant growth inhibition in E. coli and P. aeruginosa, and combination with anticancer drug geldanamycin (2µg/mL) showed selective growth inhibition against P. aeruginosa and S. aureus. Three major compounds, i.e., eugenol, 3-hexene-ol and stigmasterol, were primarily identified from Piper betel leaf extractions. Among the individual compounds, eugenol treatment showed improved growth inhibition compared with stigmasterol and 3-hexene-ol. CONCLUSIONS We are reporting potential anti-bacterial compounds from Piper betel against both gram-positive and gram-negative bacteria either alone or in combination with drug treatment.

Diferuloylmethane augments the cytotoxic effects of piplartine isolated from Piper chaba.

Natural compound based anticancer drug discovery is gaining interest against a wide variety of tumors. E-piplartine (trans-piplartine), a natural compound isolated from Piper chaba roots is examined against rat histiocytoma (BC-8), mouse embryonal carcinoma (PCC4), mouse macrophages (P388D1 and J774), and human neuroblastoma (IMR32) tumor cells. While Z-piplartine (cis-piplartine) failed to induce cytotoxicity (even at higher concentrations, 50 microM), E-piplartine induced a dose-dependent cytotoxicity (2-24 microM) in different tumor cells. The combinatorial treatment of piplartine with diferuloylmethane (curcumin), an anti-inflammatory and anticancer agent, significantly enhanced the piplartine induced cytotoxicity in tumor cells. Diferuloylmethane itself is not cytotoxic at 15 microM concentration; however, potentiated the piplartine induced cytotoxicity. The tumor cell killing with piplartine is preceded by G1 cell cycle arrest, and surpassed diferuloylmethane induced G2/M arrest when used in combination. In PCC4 cells, piplartine inhibited the cell cycle progression by inactivating cdk2 and destabilizing cyclin D1, whereas diferuloylmethane combination inhibited the ERK1/2 and Raf-1 signaling in addition to the inhibition of cell cycle progression. The over expression of heat shock protein 70, Hsp70 in rat histiocytic tumor cells interfered with piplartine induced cytotoxicity, hence, a cross talk between stress response and anticancer agents is presented. Our data demonstrates the biological and medicinal importance of piplartine isolated from the roots of P. chaba, and indicates that E-piplartine may be a promising candidate to use in combinatorial treatments to combat cancer.

Lack of heat shock response triggers programmed cell death in a rat histiocytic cell line

Stress response is a universal phenomenon. However, a rat histiocytic cell line, BC‐8, showed no heat shock response and failed to synthesize heat shock protein 70 (hsp70) upon heat shock at 42°C for 30 min. BC‐8 is a clone of AK‐5, a rat macrophage tumor line that is adapted to grow in culture and has the same chromosome number and tumorigenic potential as AK‐5. An increase in either the incubation temperature or time or both to BC‐8 cells leads to loss of cell viability. In addition, heat shock conditions activated apoptotic cell death in these cells as observed by cell fragmentation, formation of nuclear comets, apoptotic bodies, DNA fragmentation and activation of ICE‐like cysteine proteases. Results presented here demonstrate that BC‐8 cells cannot mount a typical heat shock response unlike all other eukaryotic cells and that in the absence of induction of hsps upon stress, these cells undergo apoptosis at 42°C.

A cross talk between cellular signalling and cellular redox state during heat-induced apoptosis in a rat histiocytoma.

Increasing evidence provides support for oxidative stress to be closely linked to apoptosis. Reactive oxygen species (ROS) are thought to be involved in many forms of programmed cell death. Though heat shock is a universal phenomenon, BC-8, a macrophage-like cell line failed to mount a typical heat shock response. In the absence of heat shock proteins and functional p53, BC-8 cells undergo apoptosis through CD95 signaling. In the present study, we have investigated the role of ROS in the regulation of apoptosis in these cells. We show that cells transfected with hsp70 and functional p53 are resistant to heat-induced apoptosis through inhibition of CD95 expression and ROS induction. Furthermore, apoptosis in BC-8 cells resulted in two bursts of ROS generation, one correlated with heat stress and intracellular depletion of GSH and the other with Bax overexpression and cytochrome c release. Antioxidants could not protect these cells from heat-induced apoptosis and the death pathway seems to be dependent on initial signaling cascade subsequently altering the intracellular redox. Hence, our data suggest that ROS generation in BC-8 cells upon heat shock is facultative but not obligatory for apoptosis.

Synthesis and anticancer effects of pongamol derivatives on mitogen signaling and cell cycle kinases

A series of oxazole and pyrazole derivatives of pongamol (1) were designed and synthesized to examine their anti-cancer activity. The cytotoxicity of these compounds was examined in three different human tumor cell lines, IMR-32, HeLa and Jurkat. Although all compounds tested were quite effective than the pongamol against all the three different types of cancer cell lines examined, the compounds (2), (5), and (6) were found to be the most active compounds of this series.Graphical abstractA series of pongamol derivatives (2–13) (oxazole and Pyrazole derivatives) have been synthesized and tested for their anti-cancer properties against the three human tumor cell lines (IMR32, HeLa, and jurkat).

Heat shock transcription factors regulate heat induced cell death in a rat histiocytoma.

Heat shock response is associated with the synthesis of heat shock proteins (Hsps) which is strictly regulated by different members of heat shock transcription factors (HSFs). We previously reported that a rat histiocytoma, BC-8 failed to synthesize Hsps when subjected to typical heat shock conditions (42°C, 60 min). The lack of Hsp synthesis in these cells was due to a failure in HSF1 DNA binding activity. In the present study we report that BC-8 tumor cells when subjected to heat shock at higher temperature (43°C, 60 min) or incubation for longer time at 42°C, exhibited necrosis characteristics; however, under mild heat shock (42°C, 30 min) conditions cells showed activation of autophagy. Mild heat shock treatment induced proteolysis of HSF1, and under similar conditions we observed an increase in HSF2 expression followed by its enhanced DNA binding activity. Inhibiting HSF1 proteolysis by reversible proteasome inhibition failed to inhibit heat shock induced autophagy. Compromising HSF2 expression but not HSF1 resulted in the inhibition of autophagy, suggesting HSF2 dependent activation of autophagy. We are reporting for the first time that HSF2 is heat inducible and functions in heat shock induced autophagic cell death in BC-8 tumor cells.

Heat induced expression of CD95 and its correlation with the activation of apoptosis upon heat shock in rat histiocytic tumor cells

The heat shock response is a universal phenomenon and is among the most highly conserved cellular responses. However, BC‐8, a rat histiocytoma, fails to mount a heat shock response unlike all other eukaryotic cells. In the absence of induction of heat shock proteins, apoptotic cell death is activated in BC‐8 tumor cells upon heat shock. We demonstrate here that stable transformants of BC‐8 tumor cells transfected with hsp70 cDNA constitutively express hsp70 protein and are transiently protected from heat induced apoptosis for 6–8 h. In addition heat stress induces CD95 gene expression in these tumor cells. There is a delay in CD95 expression in hsp70 transfected cells suggesting a correlation between the cell surface expression of CD95 and the time of induction of apoptosis in this tumor cell line. Also expression of CD95 antigen appears to inhibit the interaction between heat shock factors and heat shock elements in these cells resulting in the lack of heat shock response.