A histidine kinase PmHHK1 regulates polar growth, sporulation and cell wall composition in the dimorphic fungus Penicillium marneffei.

Penicillium marneffei is an important opportunistic dimorphic fungal pathogen that can cause fatal systemic mycosis in AIDS patients. To find new ways of overcoming infection, candidate virulence associated genes and virulence mechanisms are under intensive investigation. In the present study, we have examined the function of a novel P. marneffei histidine kinase gene (PmHHK1) using dsRNAi mediated by Agrobacterium tumefaciens. Our results showed that reduction of PmHHK1 expression produces significant changes in morphogenesis (including polarized growth), sporulation and cell wall composition. Two-component signaling systems are widespread in the eukaryotes outside the animal kingdom, and could be potential drug targets for antifungal chemotherapy.

Single-walled carbon nanohorn aggregates promotes mitochondrial dysfunction-induced apoptosis in hepatoblastoma cells by targeting SIRT3.

Single-walled carbon nanohorns (SWNHs) can accumulate in a variety of cell types or tissues and exert biological effects, which have been demonstrated to induce apoptosis in hepatoblastoma cells. However, the role and molecular mechanisms of SWNHs remain unclear. The mitochondrion is an important subcellular structure and may contribute to apoptosis that is induced by SWNHs in hepatoblastoma cells. To address this question, the mitochondrial function of HepG2 or L02 cells that were treated with SWNHs was examined. The results indicated that SWNHs were able to decrease the mitochondrial membrane potential and suppress the activity of the Na+/K+-ATPase. Secondly, HepG2 cells and L02 cells were treated with SWNHs in vivo and in vitro. The expression of mitochondrial-associated proteins [acyl-CoA synthetase short chain family member 1, Bax, cytochrome C (CYT-C), sodium channel epithelial 1α subunit, sirtuin 3 (SIRT3) and voltage-dependent anion channel 1] was analyzed by western blotting and immunohistochemical staining. The results revealed that SWNH treatment was able to alter the expression of multiple mitochondrial apoptotic pathway-associated proteins in HepG2 cells. SWNH treatment was able upregulate the expression of SIRT3, CYT-C and VDAC1 and downregulate the expression of AceCS2, but it had a more stable effect on SIRT3. However, similar findings were not observed in L02 cells. Therefore, the data from the present study indicated that SWNHs might be used as a safe anticancer agent, where it is able to trigger mitochondrial dysfunction-induced apoptosis by upregulating SIRT3 expression in HepG2 cells.

Titanium dioxide nanoparticles induce mitochondria-associated apoptosis in HepG2 cells.

Widespread applications of nanosized materials over the past decade have prompted investigations of desirable properties and potential hazards to humans and the environment. Titanium dioxide (TiO2) nanoparticles are one of the most widely used nanoparticles. To investigate the effect of biological functions induced by TiO2 nanoparticles (10 nm: TiO2 NPs) on human liver cell lines, normal liver cell line L02 and hepatoma cell line HepG2 were co-cultured with exogenous TiO2 NPs. Cell growth and proliferation, cell cycle, and the apoptosis rate were analyzed. The effects of TiO2 NPs on the expression levels of apoptosis-associated protein caspase-3 and the membrane channel protein αENaC and caspase-3/7 activity were determined. Moreover, the influence of TiO2 NPs on the expression levels of the mitochondria-related proteins SIRT3, VDAC1, and ACSS1, the mitochondrial membrane potential and the ADP/ATP ratio were also examined. Our results revealed that TiO2 NPs inhibited the growth and proliferation of HepG2 cells, suppressed the S phase of cell cycling, and induced apoptosis of HepG2 cells. Following an increase in concentration, the inhibitory effect induced by TiO2 NPs on proliferation and cell cycle was more evident, and the apoptosis rate increased in a significant concentration-dependent manner, whereas there was no significant effect on the growth, proliferation, apoptosis, and cell cycle of L02 cells. In addition, the results of western blot showed that in HepG2 cells, TiO2 NPs upregulated the expressions of the apoptosis-related protein caspase-3 and the membrane channel protein αENaC in a concentration-dependent manner. However, in L02 cells, there was no significant difference in the expression levels of caspase-3 or αENaC. Furthermore, TiO2 NPs induced depolarization of the mitochondrial membrane, upregulated the expression levels of the mitochondria-related proteins SIRT3 and VDAC1, and downregulated the expression level of the key respiratory chain protein ACSS1 in HepG2 cells. However, in L02 cells, the expressions of SIRT3, VDAC1, and ACSS1 exhibited no clear change. The apoptosis of HepG2 cells induced by TiO2 NPs may be achieved by regulating intracellular osmotic pressure; moreover, upregulating the expression of the channel protein αENaC or the mitochondrial porin VDAC1 and depolarizing the mitochondrial membrane of HepG2 cells resulted in the loss of Cyt-c and ATP and further activated caspase-3. To further confirm the above results, a nude mouse xenograft model was employed. After a certain period of treatment with TiO2 NPs, the nude mice were sacrificed, tumors were removed, and the expression of related proteins was detected. Immunohistochemistry and western blot results showed that the expressions of the proteins VDAC1 and SIRT3 were clearly upregulated in tissues treated to TiO2 NPs, whereas the expression of ACSS1 was downregulated. The results were consistent with the above in vitro results. All the above results confirmed that TiO2 NPs can act as a safe antitumor agent.

Chaetocin induces cell cycle arrest and apoptosis by regulating the ROS-mediated ASK-1/JNK signaling pathways.

The present study demonstrated that chaetocin, a natural small-molecule product produced by Chaetomium fungal species and a potential anticancer agent, inhibited the viability and invasive ability of the human intrahepatic cholangio-carcinoma cell line CCLP-1 in vivo and in vitro as revealed by CCK-8 and Transwell invasion assays and mouse xenograft tumor experiments. As determined using flow cytometry and intracellular ROS assays, chaetocin was found to induce cell cycle arrest and oxidative stress, leading to CCLP-1 cell apoptosis. Cell apoptosis can be initiated via different apoptotic signaling pathways under oxidative stress. As determined by western blot analysis, expression levels of the apoptosis signal-regulating kinase 1 (ASK-1) signalosome and its downstream c-Jun N-terminal kinase (JNK) signaling pathway were increased under oxidative stress stimulation. These findings indicate that chaetocin arrests the cell cycle and induces apoptosis by regulating the reactive oxygen species-mediated ASK-1/JNK signaling pathways.

Histone deacetylase inhibitor screening identifies HC toxin as the most effective in intrahepatic cholangiocarcinoma cells.

Histone deacetylases (HDACs) are highly expressed in intrahepatic cholangiocarcinoma (ICC) and are associated with poor prognosis of these patients. The aim of the present study was to explore the inhibitory effects of HDAC inhibitors on ICC cells and identify effective and sensitive drugs for ICC. Effects of 34 HDAC inhibitors were screened through two rounds of cell viability assays, and HC toxin, a cyclic tetrapeptide first isolated from the secondary metabolite of Helminthosporium carbonum, exhibited an antitumor activity superior to that of the other HDAC inhibitors and gemcitabine. The mechanisms involved in the inhibitory effects of HC toxin on CCLP-1 cells were investigated by cell counting, colony formation assay, cell morphological observation, real-time PCR, western blotting and flow cytometry. It was demonstrated that HC toxin inhibited the cell proliferation and clone formation ability of the CCLP-1 cells. HC toxin increased the acetyl-histone H4 level and this was associated with the inhibitory effect of HC toxin on the CCLP-1 cells. We also found that HC toxin reduced the level of HDAC1 protein in a post-transcriptional manner. Morphological observation showed multiple morphological changes and indicated the possibility of cell differentiation owing to HC toxin. With increasing concentration of HC toxin, the cell cycle was gradually arrested at the G0/G1 stage and the percentage of apoptotic cells increased which was not mainly through the caspase-3-dependent ways. These results indicated that HC toxin was the most effective among the various HDAC inhibitors with multiple functions in the suppression of ICC in vitro. Thus, HC may be a potential chemotherapeutic for ICC.

Effects of deep low temperature on secondary branches enclosed in Glisson's system of pig hepatic lobes.

BACKGROUND: Hepatic cryosurgery, a safe and effective approach for an unresectable hepatoma, has been extensively applied in clinical setting. But whether direct deep freezing to the tumor involving special location, i.e., Glisson's system of certain hepatic lobe could cause the impairment of the duct system or post impairment effect remains a mystery. Consequently, the aim of the study was to investigate the effect of freezing on the secondary branches enclosed in the Glisson's system of certain hepatic lobes. METHODS: Twenty pigs were divided into 2 groups randomly. The treated group: the area of secondary branches of the Glisson's system in the left lateral lobe was frozen by a cryoprobe at deep low temperature for 3 minutes, with the blockage of blood flow from the porta hepatis. The control group: only the blockage of the porta hepatis was performed for 3 minutes. Serologic examinations, color Doppler examination and pathological observation were used for evaluation of this procedure postoperatively. RESULTS: The frozen hepatic parenchyma, the wall of the frozen secondary bile duct and portal vein showed necrosis. However, the frozen hepatic artery ramification did not show any obvious changes postoperatively. Eight weeks after cryosurgery, the lumen of the hepatic artery and portal vein maintained unobstructed. Meanwhile, atrophy and fibroplasia occurred in the related hepatic lobe. All the animals recovered well. CONCLUSIONS: Animals could tolerate the direct deep-freezing of the area of secondary branches enclosed in the Glisson' s system. The therapeutic effect could be attained by the necrosis of the frozen hepatic parenchyma.

Relationship of changes in cognitive and depressive symptoms during antidepressant treatment of individuals with geriatric depression and their relationship to the APOE epsilon 4 allele.

BACKGROUND: Co-occurring cognitive impairment in geriatric depression may not improve with antidepressant treatment and it may progress to dementia. AIM: Assess the relationship between changes in cognitive and depressive symptoms among patients with geriatric depression and their association with the APOE epsilon 4 allele before and after antidepressant treatment. METHODS: The presence of the APOE epsilon 4 allele was assessed in 64 incident cases of geriatric depression and 31 elderly individuals without depression and the Geriatric Depression Scale (GDS), Mini-Mental State Examination (MMSE), digit span test, and Trail Making Tests A and B (TMT-A, TMT-B) were administered to these subjects at baseline and 12 months after baseline, during which time the depressed group received standardized treatment with selective serotonin reuptake inhibitors (SSRIs). RESULTS: Prior to treatment patients with geriatric depression had significantly worse cognitive functioning than control subjects and 31 (48%) met criteria for mild cognitive impairment (MCI). After treatment depressed patients with and without comorbid MCI both had significant improvements in their depressive and cognitive symptoms, but those with MCI had more residual symptoms. The severity of cognitive symptoms was not associated with the severity of depressive symptoms at baseline, but they were positively correlated at the 12-month follow-up. The APOE epsilon 4 allele was identified in 14% (9/64) of the patients and in 3% (1/31) of the controls (Fisher's Exact Test, p=0.158). Compared to depressed patients without the allele, depressed patients with the allele had more severe cognitive deficits both before and after treatment, though only some of these differences were statistically significant. CONCLUSIONS: There is substantial cognitive impairment in elderly individuals with geriatric depression. Both the depressive and cognitive symptoms improve with standard SSRI treatment, but individuals with comorbid MCI have more residual depressive and cognitive symptoms after treatment. The APOE epsilon 4 allele is associated with greater cognitive impairment in geriatric depressed patients and may be associated with less responsiveness of cognitive symptoms to antidepressant treatment.