Heat Killed Attenuated Leishmania Induces Apoptosis of HepG2 Cells Through ROS Mediated p53 Dependent Mitochondrial Pathway

Cytotoxic effect of attenuated Leishmania on liver cancer cells by inducing ROS generation. Methods: Spectrophotometric study to analyze cell death and levels of different active caspases. Flow cytometric study was done to analyze apoptosis induction and ROS generation and levels of different protein. Western blot analysis was performed to study the levels of protein. Confocal microscopy was done to ascertain the expression of different apoptotic markers. Results: We have now observed that attenuated Leishmania donovani UR6 also has potentiality towards growth inhibition of HepG2 cells and investigated the mechanism of action. The effect is associated with increased DNA fragmentation, rise in number of annexinV positive cells, and cell cycle arrest at G1 phase. The detection of unregulated levels of active PARP, cleaved caspases 3 and 9, cytosolic cytochrome C, Bax, and Bad, along with the observed downregulation of Bcl-2 and loss of mitochondrial membrane potential suggested the involvement of mitochondrial pathway. Enhanced ROS and p53 levels regulate the apoptosis of HepG2 cells. NAC was found to inhibit p53 production but PFT-α has no effect on ROS generation. In conclusion, Leishmania donovani UR6 efficiently induces apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. Conclusion: It has been reported earlier that some parasites show prominent cytotoxic effect and prevent tumor growth. From our study we found that Leishmania donovani UR6 efficiently induced apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. This study has rejuvenated the age old idea of bio-therap

Heat Killed Attenuated Leishmania Induces Apoptosis of HepG2 Cells Through ROS Mediated p53 Dependent Mitochondrial Pathway

Background/Aims: Cytotoxic effect of attenuated Leishmania on liver cancer cells by inducing ROS generation. Methods: Spectrophotometric study to analyze cell death and levels of different active caspases. Flow cytometric study was done to analyze apoptosis induction and ROS generation and levels of different protein. Western blot analysis was performed to study the levels of protein. Confocal microscopy was done to ascertain the expression of different apoptotic markers. Results: We have now observed that attenuated Leishmania donovani UR6 also has potentiality towards growth inhibition of HepG2 cells and investigated the mechanism of action. The effect is associated with increased DNA fragmentation, rise in number of annexinV positive cells, and cell cycle arrest at G1 phase. The detection of unregulated levels of active PARP, cleaved caspases 3 and 9, cytosolic cytochrome C, Bax, and Bad, along with the observed downregulation of Bcl-2 and loss of mitochondrial membrane potential suggested the involvement of mitochondrial pathway. Enhanced ROS and p53 levels regulate the apoptosis of HepG2 cells. NAC was found to inhibit p53 production but PFT-α has no effect on ROS generation. In conclusion, Leishmania donovani UR6 efficiently induces apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. Conclusion: It has been reported earlier that some parasites show prominent cytotoxic effect and prevent tumor growth. From our study we found that Leishmania donovani UR6 efficiently induced apoptosis in HepG2 cells through ROS mediated p53 dependent mitochondrial pathway. This study has rejuvenated the age old idea of bio-therapy

MicroRNAs: As Critical Regulators of Tumor- Associated Macrophages

Emerging shreds of evidence suggest that tumor-associated macrophages (TAMs) modulate various hallmarks of cancer during tumor progression. Tumor microenvironment (TME) prime TAMs to execute important roles in cancer development and progression, including angiogenesis, matrix metalloproteinases (MMPs) secretion, and extracellular matrix (ECM) disruption. MicroRNAs (miRNAs) are critical epigenetic regulators, which modulate various functions in diverse types of cells, including macrophages associated with TME. In this review article, we provide an update on miRNAs regulating differentiation, maturation, activation, polarization, and recruitment of macrophages in the TME. Furthermore, extracellular miRNAs are secreted from cancerous cells, which control macrophages phenotypic plasticity to support tumor growth. In return, TAMs also secrete various miRNAs that regulate tumor growth. Herein, we also describe the recent updates on the molecular connection between tumor cells and macrophages. A better understanding of the interaction between miRNAs and TAMs will provide new pharmacological targets to combat cancer

Analysis of apoptosis and cell cycle arrest in U937 cells by flow cytometry.

<p>Cells were treated with 150 μg/ml of pLLD and binding of Annexin-V/FITC to phosphatidyl serine was measured by flow cytometry to determine the percentages of apoptotic cells in time dependent manner (A). Study of cell cycle arrest in U937 cells was carried out by propidium iodide staining. Percentage of G0/G1 cell population increases after treatment of 150 μg/ml of pLLD in time dependent manner (B). The data are represented as mean ± SEM from triplicate independent experiments (*P>0.05; ** P>0.01).</p

TLC profile of pLLD (A). Growth inhibitory effect of pLLD on different cancer cells including U937, K562, HL-60 and MOLT-4 at different concentrations (0, 25, 50, 150, 200 μg/ml) at 24 h; viability was measured by MTT assay (B). The data are represented as mean ± SEM from triplicate independent experiments. (*P>0.05; ** P>0.01) Viability of U937 cells was measured in time dependent manner by flow cytometry after treatment with pLLD at the concentration 150 μg/ml (C). Morphological and nuclear changes observed in U937 cells after treatment of 150 μg/ml pLLD for 24 h (D). Morphological changes were seen under light microscope, and nuclear changes (Right to left) under fluorescence microscope after DAPI and A.O./EtBr staining respectively.

<p>TLC profile of pLLD (A). Growth inhibitory effect of pLLD on different cancer cells including U937, K562, HL-60 and MOLT-4 at different concentrations (0, 25, 50, 150, 200 μg/ml) at 24 h; viability was measured by MTT assay (B). The data are represented as mean ± SEM from triplicate independent experiments. (*P>0.05; ** P>0.01) Viability of U937 cells was measured in time dependent manner by flow cytometry after treatment with pLLD at the concentration 150 μg/ml (C). Morphological and nuclear changes observed in U937 cells after treatment of 150 μg/ml pLLD for 24 h (D). Morphological changes were seen under light microscope, and nuclear changes (Right to left) under fluorescence microscope after DAPI and A.O./EtBr staining respectively.</p

ROS generation in U937 cells upon treatment with pLLD (150 μg/ml).

<p>Flow cytometric analysis of ROS by DCFDA showed the degrees of scavenging effect in time dependent manner (A). Evaluation of mitochondrial membrane potential using JC-1 showed time dependent changes (B). Assessment of activation of caspases 9 and 3 with inhibitors and of DNA fragmentation by pLLD was done in a time dependent analysis by ELISA (C, D, E and F, respectively). Expression of pro- and anti-apoptotic molecules was assessed by western blot (G). Analysis of cytochrome C activation upon pLLD treatment was performed by confocal microscopy (H). The data are reported as the mean ± SEM of triplicate experiments (*P>0.05; ** P>0.01; *** P>0.001).</p

The effect of pLLD on MAPK pathway in U937 cells.

<p>Representative western blot results show phospho-ERK1/2/ERK1/2, phospho-JNK/JNK and phospho-p38/p38 expressions in U937 cells after incubation with pLLD (A). AP-1 expression in U937 cells 24 h after treatment of pLLD, evaluated by confocal microscopy (B) (*P>0.05; ** P>0.01; *** P>0.001).</p

Cytotoxic activity and apoptosis-inducing potential of di-spiropyrrolidino and di-spiropyrrolizidino oxindole andrographolide derivatives.

Anticancer role of andrographolide is well documented. To find novel potent derivatives with improved cytotoxicity than andrographolide on cancer cells, two series of di-spiropyrrolidino- and di-spiropyrrolizidino oxindole andrographolide derivatives prepared by cyclo-addition of azomethine ylide along with sarcosine or proline (viz. sarcosine and proline series respectively) and substitution of different functional groups (-CH3, -OCH3 and halogens) were examined for their cytotoxic effect on a panel of six human cancer cell lines (colorectal carcinoma HCT116 cells, pancreatic carcinoma MiaPaCa-2 cells, hepatocarcinoma HepG2 cells, cervical carcinoma HeLa cells, lung carcinoma A549 and melanoma A375 cells). Except halogen substituted derivatives of proline series (viz. CY2, CY14 and CY15 for Br, Cl and I substitution respectively), none of the other derivatives showed improved cytotoxicity than andrographolide in the cancer cell lines examined. Order of cytotoxicity of the potent compounds is CY2>CY14>CY15>andrographolide. Higher toxicity was observed in HCT116, MiaPaCa-2 and HepG2 cells. CY2, induced death of HCT116 (GI50 10.5), MiaPaCa-2 (GI50 11.2) and HepG2 (GI50 16.6) cells were associated with cell rounding, nuclear fragmentation and increased percentage of apoptotic cells, cell cycle arrest at G1 phase, ROS generation, and involvement of mitochondrial pathway. Upregulation of Bax, Bad, p53, caspases-3,-9 and cleaved PARP; downregulation of Bcl-2, cytosolic NF-κB p65, PI3K and p-Akt; translocation of P53/P21, NF-κB p65 were seen in CY2 treated HCT116 cells. Thus, three halogenated di-spiropyrrolizidino oxindole derivatives of andrographolide are found to be more cytotoxic than andrographolide in some cancer cells. The most potent derivative, CY2 induced death of the cancer cells involves ROS dependent mitochondrial pathway like andrographolide