scholarly journals Metformin and Calcitriol Enhance 5-Fluorouracil Tumoricidal Effects in Colon Cancer By Modulating The PI3K/Akt/PTEN/mTOR Network

2022 ◽  
Author(s):  
Riyad Almaimani ◽  
Akhmed Aslam ◽  
Jawwad Ahmad ◽  
Mahmoud Zaki El-Readi ◽  
Mohamed El-Boshy ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cytochrome C ◽  
Cell Lines ◽  
Caspase 3 ◽  
G1 Phase ◽  
Proliferation Markers ◽  
Sw620 Cell ◽  
Cell Numbers ◽  
Anti Cancer

Abstract Purpose: Chemoresistance to 5-Fluorouracil (5-FU) is common during colorectal cancer (CRC) treatment. This study measured the chemotherapeutic effects of 5-FU, calcitriol, and/or metformin single/dual/triple regimens as complementary/alternative therapies. Methods: Ninety male mice were divided into: negative and positive (PC) controls, 5-FU, Cal, Met, 5-FU/Cal, 5-FU/Met, Cal/Met, and 5-FU/Cal/Met groups. Treatments lasted four weeks following CRC induction by azoxymethane. The therapeutic regimens were also applied in the SW480 and SW620 CRC cell lines. Results: The PC mice had abundant tumours, markedly elevated proliferation markers (survivin/CCND1) and PI3K/Akt/mTOR alongside reduced p21/PTEN/Cytochrome-C/Caspase-3 and apoptosis. All therapies reduced tumour numbers, with 5-FU/Cal/Met most prominent regimen. All protocols also decreased cell proliferation markers, inhibited PI3K/Akt/mTOR molecules, increased pro-apoptotic molecules with apoptosis index, and 5-FU/Cal/Met revealed the strongest anti-cancer effects. In vitro, all therapies equally induced G1-phase arrest in SW480 cells, whereas metformin-alone showed maximal SW620 cell numbers in G0/G1-phase. 5-FU/Met co-therapy also showed the highest apoptotic SW480 cell numbers (13%), whilst 5-FU/Cal/Met disclosed the lowest percentage (81%) of viable SW620 cells. Moreover, 5-FU/Cal/Met revealed maximal inhibitions of cell cycle inducers (CCND1/CCND3), cell survival (BCL2) and the PI3K/Akt/mTOR molecules alongside highest expression of cell cycle inhibitors (p21/p27), pro-apoptotic markers (BAX/Cytochrome-C/Caspase-3), and PTEN in both cell lines. Conclusions: Metformin monotherapy was superior to calcitriol, whereas the 5-FU/metformin protocol showed better anti-cancer effects relative to the other dual therapies. However, the 5-FU/Cal/Met approach displayed the best in vivo and in vitro tumoricidal effects related to cell cycle arrest and apoptosis, justifiably by enhanced modulations of the PI3K/PTEN/Akt/mTOR pathway.

scholarly journals In vitro studies of Psorinum 6x on several human cancer cell lines reveal its anti-cancer potential

2021 ◽  
Vol 14 (2) ◽  
pp. 13-14
Author(s):  
Anisur Rahman Khuda-Bukhsh
Keyword(s):  
Lung Cancer ◽  
Cell Cycle ◽  
Cell Lines ◽  
Cancer Cell ◽  
Caspase 3 ◽  
Morphological Changes ◽  
A549 Cells ◽  
Cd Spectroscopy ◽  
Anti Cancer

Objective: Psorinum therapy is claimed to combat/ameliorate a variety of human cancers, but for obvious reasons these studies are devoid of any untreated/placebo-treated controls. Therefore, if Psorinum 6x administration to cancer cells of different origin can show visible anti-cancer effects in a controlled in vitro study has been examined. Materials & Methods: Psorinum 6x was provided by Hahnemann Publishing Company (HAPCO), 165 BB Ganguly Street, Kolkata for our research. It was prepared by HAPCO by following standard homoeopathic guidelines from authentic pus cells obtained from an eczema patient being treated at National Institute of Homeopathy, Salt Lake, Kolkata. MTT assay was initially done on several cancer cell lines like A549 (lung cancer), HeLa (cervix cancer), HepG2 (liver cancer) and MCF7 (breast cancer). Psorinum 6x showed strongest anticancer effect against A549 though it also had lesser effect against other cell lines tested. Therefore, A549 was chosen as the model cell line for further study using several relevant protocols. Effects of Psorinum 6x were compared with that of "Placebo 6x" control made of the same stock of "vehicle" used for preparation of Psorinum 6x. Protocols like analysis of cell cycle progression, generation of reactive oxygen species (ROS), change in mitochondrial membrane potential (MMP) and actual cell death (apoptosis), if any, were analysed flow-cytometrically. Whether Psorinum 6x could damage DNA and induce morphological changes were also determined microscopically. Expression of different signal proteins related to cell death (apoptosis) and survival were critically studied by western blot analysis and confocal microscopy. Further, to determine if Psorinum 6x could interact directly with DNA to induce any conformational changes was also determined by circular dichroism (CD) spectroscopy. Results: Psorinum 6x treatment reduced cell viability and inhibited cell proliferation at 24h after treatment, arresting cell cycle at sub-G stage. Upon Psorinum treatment, there were increase of ROS and MMP depolarization, morphological changes and DNA damage typical of apoptosis in A549 cells, along with externalization of phosphatidyl serine. Further, an increase in p53 level, Bax translocation into mitochondria, cytochrome c release into cytosol along with reduction of Bcl2 level and caspase 3 activation were noted which eventually drove A549 cells towards apoptosis; the apoptotic signalling was found to be through mitochondria-mediated caspase 3 dependent pathway. Evidence of direct interaction of Psorinum with cellular DNA was revealed from CD-spectroscopy. Conclusion: Thus, Psorinum 6 x had positive anti-cancer effects against a number of cancer cells, of which it appeared to have strongest effect against the lung cancer cell, A549.

Design, Synthesis and In Vitro Anti-Cancer Evaluation of Novel Derivatives of 2-(2-Methyl-1,5-diaryl-1H-pyrrol-3-yl)-2-oxo-N-(pyridin-3- yl)acetamide

2020 ◽  
Vol 16 (3) ◽  
pp. 340-349
Author(s):  
Ebrahim S. Moghadam ◽  
Farhad Saravani ◽  
Ernest Hamel ◽  
Zahra Shahsavari ◽  
Mohsen Alipour ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Peripheral Neuropathy ◽  
Cell Line ◽  
Normal Cell ◽  
Caspase 3 ◽  
Annexin V ◽  
Normal Cell Line ◽  
Anti Cancer ◽  
Mcf 7

Objective: Several anti-tubulin agents were introduced for the cancer treatment so far. Despite successes in the treatment of cancer, these agents cause toxic side effects, including peripheral neuropathy. Comparing anti-tubulin agents, indibulin seemed to cause minimal peripheral neuropathy, but its poor aqueous solubility and other potential clinical problems have led to its remaining in a preclinical stage. Methods: Herein, indibulin analogues were synthesized and evaluated for their in vitro anti-cancer activity using MTT assay (on the MCF-7, T47-D, MDA-MB231 and NIH-3T3 cell lines), annexin V/PI staining assay, cell cycle analysis, anti-tubulin assay and caspase 3/7 activation assay. Results: One of the compounds, 4a, showed good anti-proliferative activity against MCF-7 cells (IC50: 7.5 μM) and low toxicity on a normal cell line (IC50 > 100 μM). All of the tested compounds showed lower cytotoxicity on normal cell line in comparison to reference compound, indibulin. In the annexin V/PI staining assay, induction of apoptosis in the MCF-7 cell line was observed. Cell cycle analysis illustrated an increasing proportion of cells in the sub-G-1 phase, consistent with an increasing proportion of apoptotic cells. No increase in G2/M cells was observed, consistent with the absence of anti-tubulin activity. A caspase 3/7 assay protocol showed that apoptosis induction by more potent compounds was due to activation of caspase 3. Conclusion: Newly synthesized compounds exerted acceptable anticancer activity and further investigation of current scaffold would be beneficial.

scholarly journals The pro-apoptosis effects of Echinacea purpurea and Cannabis sativa extracts in human lung cancer cells through caspase-dependent pathway

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Fatemeh Hosami ◽  
Azadeh Manayi ◽  
Vahid Salimi ◽  
Farshad Khodakhah ◽  
Mitra Nourbakhsh ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Cycle ◽  
Caspase 3 ◽  
Cannabis Sativa ◽  
A549 Cells ◽  
Echinacea Purpurea ◽  
G1 Phase ◽  
Cell Cycle Distribution ◽  
Cb2 Receptor ◽  
Anti Cancer

Abstract Background Considering the advantages of using medicinal herbs as supplementary treatments to sensitize conventional anti-cancer drugs, studying functional mechanisms and regulatory effects of Echinacea purpurea (as a non-cannabinoid plant) and Cannabis sativa (as a cannabinoid plant) are timely and required. The potential effects of such herbs on lung cancer cell growth, apoptosis, cell cycle distribution, cellular reactive oxygen species (ROS) level, caspase activity and their cannabinomimetic properties on the CB2 receptor are addressed in the current study. Methods The cytotoxic effect of both herb extracts on the growth of lung cancer cells (A549) was assessed using the MTT assay. The annexin-V-FITC staining and propidium iodide (PI) staining methods were applied for the detection of apoptosis and cell cycle distribution using flow cytometry. The cellular level of ROS was measured using 7′-dichlorofluorescin diacetate (DCFH-DA) as a fluorescent probe in flow cytometry. The caspase 3 activity was assessed using a colorimetric assay Kit. Results Echinacea purpurea (EP) root extract induced a considerable decrease in A549 viable cells, showing a time and dose-dependent response. The cell toxicity of EP was accompanied by induction of early apoptosis and cell accumulation at the sub G1 phase of the cell cycle. The elevation of cellular ROS level and caspase 3 activity indicate ROS-induced caspase-dependent apoptosis following the treatment of A549 cells by EP extract. The observed effects of EP extract on A549 growth and death were abrogated following blockage of CB2 using AM630, a specific antagonist of the CB2 receptor. Increasing concentrations of Cannabis sativa (CS) induced A549 cell death in a time-dependent manner, followed by induction of early apoptosis, cell cycle arrest at sub G1 phase, elevation of ROS level, and activation of caspase 3. The CB2 blockage caused attenuation of CS effects on A549 cell death which revealed consistency with the effects of EP extract on A549 cells. Conclusions The pro-apoptotic effects of EP and CS extracts on A549 cells and their possible regulatory role of CB2 activity might be attributed to metabolites of both herbs. These effects deserve receiving more attention as alternative anti-cancer agents. Graphical abstract

scholarly journals Direct Effect of Septic Plasma in Human Cell Lines Viability

2018 ◽  
Vol 47 (1-3) ◽  
pp. 270-276
Author(s):  
Grazia Maria Virzì ◽  
Chiara Borga ◽  
Chiara Pasqualin ◽  
Silvia Pastori ◽  
Alessandra Brocca ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Cell Viability ◽  
Cell Lines ◽  
Caspase 3 ◽  
Test Group ◽  
Organ Injury ◽  
Enzyme Linked Immunosorbent Assay ◽  
Caspase 9 ◽  
Renal Tubular Cells

Background: Sepsis is a life-threatening condition often associated with a high incidence of multiple organs injury. Several papers suggested the immune response by itself, with the production of humoral inflammatory mediators, is crucial in determining organ injury. However, little is known of how sepsis directly induces organ injury at the cellular levels. To assess this point, we set up an in vitro study to investigate the response of renal tubular cells (RTCs), monocytes (U937) and hepatocytes (HepG2) after 24 h-incubation with septic patients’ plasma. Methods: We enrolled 26 septic patients (“test” group). We evaluated cell viability, apoptosis and necrosis by flow cytometer. Caspase-3,-8,-9 and cytochrome-c concentrations have been analyzed using the Human enzyme-linked immunosorbent assay kit. Results: We found that a decrease of cell viability in all cell lines tested was associated to the increase of apoptosis in RTCs and U937 (p < 0.0001) and increase of necrosis in HepG2 (p < 0.5). The increase of apoptosis in RTCs and U937 cells was confirmed by higher levels of caspase-3 (p < 0.0001). We showed that apoptosis in both RTCs and U937 was triggered by the activation of the intrinsic pathway, as caspase-9 and cytochrome-c levels significantly increased (p < 0.0001), while caspase-8 did not change. This assumption was strengthened by the significant correlation of caspase-9 with both cytochrome-c (r = 0.73 for RTCs and r = 0.69 for U937) and caspase-3 (r = 0.69 for RTCs and r = 0.63 for U937). Conclusion: Humoral mediators in septic patients’ plasma induce apoptosis. This fact suggests that apoptosis inhibitors should be investigated as future strategy to reduce sepsis-induced organ damages.

In-Vitro Treatment with the AKT-Inhibitor GSK 690693 Induces Cell Death in Lymphoma Cell Lines and in Primary CLL Cells and Is Followed by Caspase-3 Activation and Cytochrome C Release.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1589-1589
Author(s):  
Dirk Winkler ◽  
Thorsten Zenz ◽  
Daniel Mertens ◽  
Annett Habermann ◽  
Hartmut Döhner ◽  
...  
Keyword(s):  
Cell Death ◽  
Cytochrome C ◽  
Conditioned Medium ◽  
Cell Lines ◽  
Caspase 3 ◽  
Spontaneous Apoptosis ◽  
Akt Inhibitor ◽  
Cytochrome C Release ◽  
Viable Cells

Abstract The PI3K/AKT pathway acts as a critical regulator of cell survival by stimulating cell proliferation and inhibiting apoptosis and has been implicated in the pathogenesis of lymphoproliferative disorders. Therefore, inhibition of AKT seems to be a highly attractive new approach for the treatment of lymphoma. We treated 9 cell lines with AKT-nhibitor (1, 10, 20 μM) over 24h and 48h respectively: EHEB (B-CLL), GRANTA-519 (MCL), JURKAT (T-ALL) BL-60, NAMALWA and BJAB (all Burkitt’s lymphoma), L363, OPM-2 and RPMI-8226 (all multiple myeloma). To determine the rates and type of AKT-inhibitor induced cell death, FACS analyses for CD19, 7AAD, active caspase-3, cytochrome c were performed. The phosphorylation status of AKT and its downstream proteins GSK3β, p70S6k and S6 was studied by Western blotting after 5–120 minutes. In addition, 11 primary CLL samples with either del 13q (n=3), del 11q (n=2), del 17p (n=3) or a normal karyotype (n=3) were treated with AKT inhibitor (10 μM; 2.5μM; 0.625 μM; 0.156 μM). CLL samples were cultured in both standard medium as well as in HS5-(human stromal cells) conditioned medium to reduce spontaneous apoptosis of CLL in-vitro. 6 out of 11 patients had unmutated VH genes. 8 Patients were untreated, 3 were previously treated. Fludarabine (0.1 μM) was added to AKT-inhibitor in 11 cases to test for synergistic effects. CLL cells were harvested after 48 hours and 5 days to measure cell viability using Celltiter-GLO-Assay. Treatment of cell lines lead to significant rates of AKT-inhibitor induced cell death (table 1), to hyperphosphorylation of AKT and to inhibition of phosphorylation of GSK3β (after 5 min) and S6 (after 20 min) in all cell lines and of p70S6k (after 120 min) in GRANTA, JURKAT, NAMALWA and BJAB. Cell death did not depend on functional p53 gene. Treatment of primary CLL samples with AKT-inhibitor alone was followed by a decrease of cell viability in a time and concentration dependent manner regardless of the medium used (table 2). Only with the lowest concentration and when cultured in HS5-conditioned medium, no further reduction of viable cells was seen between 48h and 5d. Treatment with AKT-inhibitor as a single agent seemed to be at least as effective as treatment with fludarabine. Response was independent of the genetic subgroup, VH mutation status or prior treatment. High risk cases with del 17p responded worse to fludarabine alone when compared to cases without del 17p (i.e. 75% of viable cells after 5d at 10000 μM in cases with del 17p vs. 25% in cases without del 17p). The same fludarabine resistant cases showed good responses to treatment with AKT-inhibitor (9% of viable cells after 5d at 10000 μM in cases with del 17p). A synergistic effect was not achieved by combining AKT-inhibitor and fludarabine. Culture of CLL cells in HS5-conditioned medium resulted in lower rates of spontaneous apoptosis, but also in lower rates of AKT-inhibitor induced cell death. In conclusion, in-vitro treatment with AKT-inhibitor resulted in significant rates of cell death in cell lines and primary CLL cells, even in patients with del 17p or resistance to fludarabine. In cell lines, treatment with AKT-inhibitor was followed by typical features of apoptosis such as activation of caspase-3 and cytochrome c release. In CLL samples, prior treatment did not affect in-vitro response rates. These data underline the involvement of the PI3K/Akt pathway in the pathogenesis of lymphoma and point to an efficacy of the AKT-inhibitor in the treatment of lymphoma, multiple myeloma and CLL in-vivo. Concerning CLL, the AKT-inhibitor seems to be an attractive new treatment option even for cases with high risk cytogenetics. Using HS5-conditioned medium seems to be a well functioning method to reduce spontaneous apoptosis of CLL cells in-vitro. Table 1: rates of cell death, caspase-3 activation and cytochrome c release after treatment of cell lines with AKT inhibitor (1μM, 48h) 7AAD-positive cells active caspase-3 cytochrome c release EHEB 15% − + GRANTA-519 15% + + JURKAT 17% + + BL60 24% + + NAMALWA 25% − (+) BJAB 30% + (+) L363 15% + − OPM-2 41% + + RPMI-8226 32% + (+) Table 2: mean percentage of viable cells after treatment with AKT-Inhibitor (A), fludarabine (F; 0,1μM) and their combination (A + F) measured by Celltiter-GLO-Assay 10000 nM 2500 nM 625 nM 156,25 nM 48h 5d 48h 5d 48h 5d 48h 5d A F A + F A F A + F A A F A+ F A F A+ F A HS5 + (n=8) 94 84 (n=5) 75 (n=5) 45 22 (n=5) 25 (n=5) 88 52 91 84 80 69 22 18 76 85 HS5 − (n=11) 60 79 59 8 39 21 77 27 80 79 76 28 39 34 82 (n=10) 21

Antiproliferative and Proapoptotic Effects of Akacid-Medical-Formulation on Hematologic Cell Lines.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4380-4380
Author(s):  
Hannes Neuwirt ◽  
Christina Salvador ◽  
Elisabeth Wabnig ◽  
Martin Tiefenthaler ◽  
Zoran Culig ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Caspase 3 ◽  
Flow Cytometric Analysis ◽  
G1 Phase ◽  
Caspase 8 ◽  
Antiapoptotic Proteins ◽  
Phase Arrest ◽  
Downstream Effectors ◽  
Induction Of Apoptosis

Abstract We have previously shown that Akacid-medical-formulation (AMF) exerts its antiproliferative effects on various malignant solid cell lines, including those derived from prostate via downregulation of mitogen-activated protein kinases Erk 1/2 and cell cycle regulators, e.g. cdk-2, -4, cyclin E, -D1 (Neuwirt et al. 2006). However, the effect of AMF on proliferation and induction of apoptosis in malignant hematologic cell lines has not been investigated yet. Therefore, we performed 3H-thymidine incorporation assays to assess the growth inhibition of HL-60, U-937, K-562, CEM-C7H2 cells after treatment for 48 hours with various concentrations of AMF (0.3 – 100 μM). All cell lines were dose-dependently inhibited by AMF with an IC50 of about 2.1μM. As cellular growth arrest is known to be one major reason for resistance to chemotherapeutics, we investigated the effect of AMF on the cell line CEM-C7H2-6E2, in which G1/0-phase arrest can be induced by tetracyclin-regulated expression of the cell cycle inhibitor p16INK4A (Fig. 1A). Flow cytometric analysis using Annexin-V / propidium-iodide staining showed that in G1-phase arrested cells apoptosis was induced to a similar extent (LD50 of 10μM) as compared to proliferating control cells (CEM-C7H2-2C8) (Fig. 1B). In addition, we tried to find out whether AMF induces apoptosis via the caspase-9 dependent intrinsic or the caspase-8 dependent extrinsic pathway. For this purpose, two different cell lines stably transfected with tetracyclin-responsible plasmids encoding for the antiapoptotic proteins bcl-2 (CEM-C7H2-10E1) and CrmA (CEM-C7H2-2E8) were used. After 48 hours of treatment with AMF (1 – 30μM) we found that overexpression of neither bcl-2 nor CrmA could inhibit AMF-induced apoptosis compared to control cells (LD50 15μM). Data on apoptosis obtained by flow cytometry were confirmed by Western blot analysis on activated caspase-8, -9, -3, and cleaved PARP in CEM-C7H2 cells. Caspase-8 and -9 were not activated after 48 hours. However, it is interesting that downstream effectors of apoptosis, cleaved PARP and caspase-3, were strongly induced (1000 % of control at 30μM). In conclusion, our results show a potent antiproliferative effect of AMF on leukemic cell lines. Induction of apoptosis could not be inhibited either by G1-phase arrest or overexpression of the antiapoptotic proteins bcl-2 or CrmA. Although we found a substantial activation of the downstream effectors of apoptosis by AMF including caspase-3 and PARP, the upstream pathway of activation remains unclear. Figure 1 Figure 1.

Induction of Apoptosis by Pierisin-6 in HPV Positive HeLa and HepG2 Cancer Cells is Mediated by the Caspase-3 Dependent Mitochondrial Pathway

2019 ◽  
Vol 19 (3) ◽  
pp. 337-346 ◽  
Author(s):  
Subbarayan Sarathbabu ◽  
Satheesh K. Marimuthu ◽  
Souvik Ghatak ◽  
Subramanian Vidyalakshmi ◽  
Guruswami Gurusubramanian ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cytochrome C ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Propidium Iodide ◽  
Caspase 3 ◽  
Mitochondrial Pathway ◽  
Target Cells ◽  
Specific Expression ◽  
Cycle Arrest

Background: To explore the cytotoxic and apoptotic activity of the pierisin-6 protein in HPV HeLa and HepG2 cell lines. Methods: In this study, isolation, and purification of cytotoxic Prierisin-6 from the larvae of Pieris napi by affinity column chromatography techniques. Characterization of full-length mRNA of pierisin-6 gene was performed using 3’/5’ RACE PCR. The quantitative RT-PCR used to study the developmental stage-specific expression of pierisin-6 mRNA. The most effective concentration of Pierisin-6 protein was determined by measuring cell proliferation. Apoptosis was assessed using AO/Et-Br, Propidium Iodide, and Rhodamine 123 assays, whereas protein levels of caspase 3, cytochrome C were evaluated by ELISA method. Pierisin-6 induced cell cycle arrest was determined using Propidium iodide by FACS. Results: In this study, Pierisin-6, a novel apoptotic protein was found to have cytotoxicity against HeLa, HepG2 human cancer cell lines and L-132 human lung epithelial cell line. Among the target cells, HeLa was the most sensitive to Pierisin-6. Flow cytometry analysis confirms an increased percentage of apoptotic cells in sub G1 phase and cell cycle arrest at S phase. Alteration in the transmembrane potential of mitochondria, Cytochrome c released from the mitochondrial membrane, and caspase substrate assay demonstrated the cleavage of Ac- DEVD-pNA signifying the activation of Caspase-3. These findings suggested that Pierisin-6 significantly induce apoptosis in HeLa and HepG2 cells and is attributed mainly through a mitochondrial pathway by activation of caspases. The developmental and stage-specific expression of pierisin-6 mRNA was one thousand-fold increased from second to third instar larvae and gradually declined before pupation. Conclusion: Pierisin-6 represents a promising therapeutic approach for liver cancer patients.

scholarly journals Policing Cancer: Vitamin D Arrests the Cell Cycle

2020 ◽  
Vol 21 (23) ◽  
pp. 9296
Author(s):  
Sachin Bhoora ◽  
Rivak Punchoo
Keyword(s):  
Cell Cycle ◽  
Vitamin D ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
In Vitro Studies ◽  
Vitamin D Metabolites ◽  
Cell Cycle Regulators ◽  
Cycle Arrest ◽  
Anti Cancer

Vitamin D is a steroid hormone crucial for bone mineral metabolism. In addition, vitamin D has pleiotropic actions in the body, including anti-cancer actions. These anti-cancer properties observed within in vitro studies frequently report the reduction of cell proliferation by interruption of the cell cycle by the direct alteration of cell cycle regulators which induce cell cycle arrest. The most recurrent reported mode of cell cycle arrest by vitamin D is at the G1/G0 phase of the cell cycle. This arrest is mediated by p21 and p27 upregulation, which results in suppression of cyclin D and E activity which leads to G1/G0 arrest. In addition, vitamin D treatments within in vitro cell lines have observed a reduced C-MYC expression and increased retinoblastoma protein levels that also result in G1/G0 arrest. In contrast, G2/M arrest is reported rarely within in vitro studies, and the mechanisms of this arrest are poorly described. Although the relationship of epigenetics on vitamin D metabolism is acknowledged, studies exploring a direct relationship to cell cycle perturbation is limited. In this review, we examine in vitro evidence of vitamin D and vitamin D metabolites directly influencing cell cycle regulators and inducing cell cycle arrest in cancer cell lines.

In-Vitro Treatment of Lymphoma Cell Lines with BAY 43-9006 (Sorafenib) Is Followed by Typical Features of Apoptosis and Down-Regulation of MCL-1 Pointing to an Efficacy of Sorafenib in the Teatment of Lymphoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1392-1392
Author(s):  
Dirk Winkler ◽  
Christof Schneider ◽  
Annett Habermann ◽  
Hartmut Doehner ◽  
Stephan Stilgenbauer
Keyword(s):  
Cell Cycle ◽  
Cytochrome C ◽  
Map Kinase ◽  
Cell Lines ◽  
Caspase 3 ◽  
P53 Mutation ◽  
Down Regulation ◽  
Map Kinase Pathway ◽  
Induced Apoptosis ◽  
Kinase Pathway

Abstract BAY 43-9006 (sorafenib) is a multikinase inhibitor that has shown efficacy against a variety of malignancies in preclinical models. In the treatment of lymphoma, however, its place is still to be determined. We treated 7 lymphoma cell lines with sorafenib (10μM) over 24h and 48h respectively: EHEB (B-CLL), JVM-2 (MCL), GRANTA-519 (MCL), JURKAT (T-ALL with p53 mutation), BL-60, NAMALWA and BJAB (all Burkitt’s lymphoma with del 17p and p53 mutation). To determine the rates and type of sorafenib induced apoptosis, 4-colour FACS analyses (CD19, 7AAD, active caspase-3, cytochrome c) were performed. The expression of the following proteins involved in apoptosis, cell cycle regulation and the MAP-kinase pathway was studied by Western blotting: BAX, BCL-2, MCL-1, p53, p21, p27, procaspase-3, procaspase-8, procaspase-9, PARP, ERK, JNK and p38. The posphorylation status of ERK, JNK, and p38 was investigated after 5–120 minutes of treatment. Significant rates of sorafenib induced apoptosis as detected by 7AAD-FACS were seen in EHEB (46% apoptotic rate), JVM-2 (36%), GRANTA (75%), JURKAT (85%) and BL-60 (84%) after 48 hours. Cytochrome c releases, BAX cleavage as well as a down-regulation of MCL-1 and p27 were seen in all cell lines after 48 hours independent of their p53 status (no expression of p27 was detected in JVM-2). No activation of caspase-3 was seen by FACS and no cleavage product of caspase-3 could be detected by Western blotting. However, reduced levels of procaspase-3 were detected for EHEB, BL-60, NAMALWA and JURKAT. A decrease of expression of procaspase 3/8/9 was seen in NAMALWA, BL60 and JURKAT. No regulation of caspases was seen in the MCL cell lines, despite significant rates of sorafenib induced apoptosis. In BL-60 and NAMALWA, both showing a deletion of TP53 and a p53 mutation in the remaining allele, sorafenib treatment resulted in down-regulation of all proteins studied except for BCL-2 that did not show expression change; BJAB also showed reduced levels of p21, JNK and ERK after treatment, but responded with up-regulation of p53 and BCL-2 and unchanged levels for caspases. Down-regulation of BCL-2 was only seen in GRANTA, whereas EHEB responded with an up-regulation. All cell lines except for JURKAT, the only T-cell line, exhibited declining expression of proteins of the MAP-kinase pathway. Sorafenib inhibited the posphorylation of ERK in BL60, NAMALWA and JURKAT at a concentration of 1 and 10μM after 120 minutes of treatment. No change in posphorylation status was seen in the other cell lines. Although the effects of sorafenib on caspases, cell cycle regulating proteins, downstream proteins of the MAP-kinase pathway and their posphorylation status differed among the lymphoma cell lines studied, sorafenib treatment was consistently followed by typical features of apoptosis such as BAX cleavage and cytochrome c release. Induction of cell death did not depend on functional p53 gene. Furthermore, all cell lines investigated responded with down-regulation of the anti-apoptotic protein MCL-1 and the cell cycle regulator p27. Interestingly, not all cell lines responded with activation of caspases. A central role of MCL-1 operating upstream of cytochrome c release and caspase activation as well as induction of cell death by sorafenib through both caspase-dependent and -independent pathways are in line with earlier reports on other malignancies, and point to an efficacy of sorafenib in the treatment of lymphoma.

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