Selective inhibition of histone deacetylase 6 alters the composition of circulating blood cells in a lethal septic model

Background

Phagocytes, especially monocytes, macrophages, and dendritic cells, play a pivotal role in the innate and adaptive immune responses during sepsis. We have shown that inhibition of histone deacetylase 6 improves survival and increases bacterial clearance in a mouse model of cecal ligation and puncture (CLP). The aim of this study was to determine whether this effect was associated with changes in the number and composition of different blood cell types in the circulation.

Methods

C57BL/6J mice were subjected to CLP, and 1 h later given an intraperitoneal injection of either Tubastatin A dissolved in dimethyl sulfoxide, or dimethyl sulfoxide only. Sham-operated animals were treated in an identical fashion but not subjected to CLP. Forty-eight hours later, peripheral blood was obtained via cardiac puncture and analyzed using a HemaTrue veterinary hematology analyzer.

Results

Tubastatin A administration increased the number of circulating monocytes in the sham-operated and the CLP animals. In comparison with the sham, CLP animals displayed an increase in the granulocyte percentage in white blood cells and decrease in the lymphocyte number and percentage, with a resultant increase in the granulocyte-to-lymphocyte ratio. Treatment of CLP animals with Tubastatin A decreased the granulocyte percentage and restored the lymphocyte number and percentage, which decreased the granulocyte-to-lymphocyte ratio. In the sham animals, Tubastatin A increased red blood cell number, hematocrit, and hemoglobin. This effect was not seen in CLP animals.

Conclusions

Tubastatin A treatment has significant impact on the composition of circulating blood cells. It increases the number of circulating monocytes and the red blood cell mass in sham-operated animals. In the CLP animals, it increases the monocyte count, decreases the percentage of granulocytes, restores the lymphocyte population, and decreases the granulocyte-to-lymphocyte ratio. These results may explain why Tubastatin A treatment improves survival in the septic models.     

A newly synthesized platinum-based compound (PBC-II) increases chemosensitivity of HeLa ovarian cancer cells via inhibition of autophagy

There are many mechanisms of resistance, chemoresistance of HeLa cells to anti-cancer agents seems to be autophagy-mediated. While using very effective anti-cancers such as Doxorubicin and cisplatin, cells overcome the cytotoxicity of these drugs through promotion of what so-called cytoprotective autophagy. Here in this study, we sought to introduce a novel platinum-based compound PBC-II that possesses anti-cancer activity. Our data showed that PBC-II is able to induce apoptosis at relatively low concentrations, with no detectable reactive oxygen species (ROS). However, further experiments demonstrated that exposure of HeLa cells to PBC-II did not promote autophagy; rather, it resulted in accumulation of p62 and decrease in LC3-II levels. Autophagy was then promoted in HeLa cells pharmacologically by Doxorubicin and genetically by siRNA IL-10. In order to confirm promotion of autophagy in our model, we performed acridine orange staining to assess for autophagy under microscope as well as via flow cytometry. We then measured protein level of autophagy markers p62 and LC3 by western blot. Our data indicated that PBC-II interferes with therapy-induced autophagy. We also determined PI3K activity while co-incubation of PBC-II with autophagy inducers. It was clear that PI3K activation decreased when PBC-II was co-administered with autophagy inducers. Collectively, PBC-II exerts unique anti-proliferative effects associated with inhibition of autophagy, which indicates that PBC-II is potentially a promising agent to be used in resistant ovarian tumors.     

Immunolocalization of murine type VI 3β-hydroxysteroid dehydrogenase in the adrenal gland,testis, skin,and placenta

The enzyme 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) is essential for the biosynthesis of all active steroid hormones, such as those secreted from the adrenal gland, testis, ovary, skin and placenta. The 3β-HSD enzymes exist in multiple isoforms in humans and rodents. To date, six different isoforms have been identified in the mouse, and these isoforms are speculated to play different roles in different tissues. We previously showed that the murine type VI 3β-HSD isoform (Hsd3b6) is expressed specifically in the aldosterone-producing zona glomerulosa cells within the adrenal gland and that its overexpression causes abnormally increased aldosterone synthesis, revealing a crucial (or rate-limiting) role of this enzyme in steroidogenesis. However, potential contributions of this enzyme to the steroid hormone synthesis outside the adrenal glands are poorly understood. This paucity of knowledge is partly because of the lack of isoform-specific antibody that can be used for immunohistochemistry. Here, we report the development and characterization of specific antibody to Hsd3b6 and show the results of immunohistochemistry for the adrenal gland, testis, ovary, skin and placenta. As expected, Hsd3b6 immunoreactivities within the adrenal gland were essentially confined to the zona glomerulosa cells, where aldosterone is produced. By contrast, no immunopositive cells were observed in the zona fasciculata, which is where corticosterone is produced. In the gonads, while the ovaries did not show any detectable immunoreactivity to Hsd3b6, the testes displayed intense immunoreactivities within the interstitial Leydig cells, where testosterone is produced. In the skin, positive immunoreactivities to Hsd3b6 were only seen in the sebaceous glands, suggesting a specific role of this enzyme in sebaceous function. Moreover, in the placenta, Hsd3b6 was specifically found in the giant trophoblast cells surrounding the embryonic cavity, which suggests a role for this enzyme in local progesterone production that is required for proper embryonic implantation and/or maintenance of pregnancy. Taken together, our data revealed that Hsd3b6 is localized in multiple specific tissues and cell types, perhaps thereby involved in biosynthesis of a number of tissue-specific steroid hormones with different physiological roles.