Synergistic effects of isomorellin and forbesione with doxorubicin on apoptosis induction in human cholangiocarcinoma cell lines

Abstract Background: The methylation inhibitor decitabine (DAC) has great therapeutic value for myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, DAC monotherapy is associated with relatively low rates for overall response and complete remission. We aimed to investigate the effects of the combination of DAC and homoharringtonine (HHT) in the SKM-1 and Kg-1a cell lines and explore their associated mechanisms. Methods: Cell viability was estimated by MTS assay. Cell apoptosis were detected by flow cytometry analysis and PI/Annexin V staining. Western blot and quantitative reverse transcription-polymerase chain reaction assays were performed to detect the expression of apoptosis-related genes including caspase-3, caspase-9, BCL-2 and BCL-XL and DNA methyltransferases(DNMT) including DNMT1, DNMT3A and DNMT3B. Changes in LINE-1 gene methylation were assessed by quantitative polymerase chain reaction after bisulfite conversion. The effects of the combinations were estimated using CalcuSyn software. Results: The combination of DAC and HHT showed synergistic effects for inhibiting cell viability in SKM-1 and KG-1a cell lines. This combination can enhance inhibition of colony formation and apoptosis induction of DAC alone in SKM-1 cell line. However, in Kg-1a cells, this combination only enhanced the apoptosis induction of DAC alone. For SKM-1 cells, further study found that different doses of DAC and HHT alone have different effects on the expression of the apoptosis-related genes caspase-3, caspase-9, BCL-XL and BCL-2. The combination of 0.4 μM DAC and 5 nM HHT treatment significantly increased the mRNA expression of caspase-3 (P=0.0005) and caspase-9 (P=0.0075) and decreased the expression of BCL-2 (P=0.0331), but has no significantly effect on the BCL-XL (P=0.3436) compared with 0.4 μM DAC alone. However, 4 μM DAC plus 50 nM HHT had no significant effects on the mRNA and protein expression of the apoptosis-related genes examined compared with 4 μM DAC alone (P>0.05). Low-dose DAC induced greater hypomethylation than higher doses of the drug. Whereas HHT had no demethylation effects, and also had no enhancement effects with DAC on the hypomethylation and mRNA and protein expression of DNMT1, DNMT3A and DNMT3B in SKM-1 cells. Conclusion: The combination of DAC and HHT has synergistic effects on cell viability inhibition and enhancement effects on cell apoptosis in SKM-1 and KG-1a cell lines. But this combination did not enhance the hypomethylation effect of DAC.These data suggest that DAC used in combination with HHT has clinical potential in the treatment of MDS and AML. Disclosures No relevant conflicts of interest to declare.

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Bcl-2 and mTOR as Effective Targets for Molecular Therapy of Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v116.21.3228.3228 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3228-3228

Author(s):

Stefano Iacovelli ◽

Maria Rosaria Ricciardi ◽

Andrea Miele ◽

Paola Bergamo ◽

Roberto Licchetta ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Growth Inhibition ◽

Cell Line ◽

Cell Lines ◽

Synergistic Effects ◽

Lymphoblastic Leukemia ◽

Mtor Inhibitors ◽

Primary Cells ◽

Apoptosis Induction ◽

Combined Use

Abstract Abstract 3228 Although they frequently achieve complete remission (CR), adult patients with acute lymphoblastic leukemia (ALL) subsequently experience leukemia relapse, which represents an unresolved therapeutic problem. Based on the observation that ALL cells are frequently characterized by the deregulation of the apoptotic machinery, we and others have evaluated pre-clinically the activity of ABT-737 (kindly provided by Abbott Laboratories), a BH3-mimetic Bcl-2/Bcl-XL inhibitor, displaying a potent growth-inhibitory activity in ALL cell lines and primary cells. However ABT-737 binds to the anti-apoptotic protein Mcl-1 with low affinity and Mcl-1 expression may mediate resistance to ABT-737. Since ALL is also characterized by the aberrant activation of the mTOR and related signalling pathways, in the present study we further evaluated the combined Bcl-2/Bcl-XL (by ABT-737) and mTOR (by CCI-779) inhibition focusing, in particular, on the activity of combined molecularly targeted therapies on resistant cells. In MOLT-4 cells, ABT-737 induced dose and time-dependent growth inhibition (IC-50= 198nM) followed, at higher concentrations (250-500nM), by apoptosis induction. In contrast, the CEM-S, CEM-R, JURKAT, DAUDI and RAJI cells proved resistant (IC-50 >5 μM). When we explored the effects of CCI-779 on the aforementioned cell lines, only minor cytostatic effects were observed (IC-50 0.5–28.2μM). MOLT-4 cells, for example, showed a flat dose-response curve (35-55% growth inhibition) at concentrations ranging between 1 and 5000 nM (IC50=9,87μM) and apoptosis induction was not seen until 5000 nM. We next investigated the effects of the combined use of ABT-737 and CCI-779 (each at 1000nM) in the ABT-resistant JURKAT cells. A significant (p= 0.04) induction of apoptosis was observed with the combination, as compared with single agents, after 24 h (47.7% ±5.9 of cells with sub-G1 DNA content with ABT-737 + CCI-779, compared to 17.4% ±1.5 and 4.2% ±1.5 with ABT-737 and CCI-779 as single agents, respectively). Similarly, when we exposed CEM-R cells to the drug combination (ABT-737 1000nM and CCI-779 5000nM) for 24 h, a strikingly stronger apoptosis induction (sub-G1 peak= 75.3% ±16.8) was observed, compared to single agents (15.8% ±7.2 and 4.2% ±1.9 with ABT-737 and CCI-779 alone, respectively) (p=0.0003). These effects were confirmed by measuring Annexin V binding. WB analysis showed decreased Mcl-1 levels, following exposure to CCI-779 and further downregulation in response to combined ABT-737+CCI-779 in the CEM-R cell line. These effects, however, were not seen in the parental CEM-S cell line. Primary cells, obtained from 10 ALL patients, showed an increase of the sub-G1 peak in 7/10 and in 4/10 samples, after exposure to ABT-737 (50nM) and CCI-779 (5000 nM), while synergistic effects on apoptosis induction were observed in 4/10 samples after exposure to the combination. In summary, we observed that the combined use of Bcl-2/Bcl-XL and mTOR inhibitors may exert synergistic cytotoxic effects in some resistant ALL models and this effect is associated with CCI-779-induced Mcl-1 down-regulation. Synergistic effects between these inhibitors were also found in a proportion of primary ALL samples, thus supporting further studies of combined Bcl-2/Bcl-XL and mTOR inhibitors, to overcome ALL resistance. Disclosures: Petrucci: Celgene: Honoraria; Janssen Cilag: Honoraria. Tafuri:Sigma-Tau: Research Funding.

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The synergistic effects of valproic acid and fluvastatin on apoptosis induction in glioblastoma multiforme cell lines

The International Journal of Biochemistry & Cell Biology ◽

10.1016/j.biocel.2017.10.003 ◽

2017 ◽

Vol 92 ◽

pp. 155-163 ◽

Cited By ~ 9

Author(s):

Yung-Lung Chang ◽

Li-Chun Huang ◽

Ying-Chuan Chen ◽

Yi-Wen Wang ◽

Dueng-Yuan Hueng ◽

...

Keyword(s):

Valproic Acid ◽

Glioblastoma Multiforme ◽

Cell Lines ◽

Synergistic Effects ◽

Apoptosis Induction

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In Vitro Evaluation of Chitosan Induced Cytotoxicity against Cancerous Cell lines: MCF-7, Saos-2 and HeLa

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520619666190507113949 ◽

2019 ◽

Vol 19 ◽

Author(s):

Zeinab Abedian ◽

Niloofar Jenabian ◽

Ali Akbar Moghadamnia ◽

Ebrahim Zabihi ◽

Roghayeh Pourbagher ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Lines ◽

Anticancer Agents ◽

Fibroblast Cells ◽

Apoptosis Induction ◽

Cytotoxic Effects ◽

Cancerous Cell ◽

Significant Concentration ◽

Mcf 7

Objective/ Background: Cancer is still the most common cause of morbidity in world and new powerful anticancer agents without severe side effects from natural sources is important. Methods: The evaluation of cytotoxicity and apoptosis induction was carried out in MCF-7,HeLa and Saos-2 as cancerous cell lines with different histological origin and human fibroblast served as control normal cell. The cells were treated with different concentrations of chitosan and the cytotoxicity was determined using MTT assay after 24, 48 and 72 h .The mode of death was evaluated by flow cytometry . Results: While both types of chitosan showed significant concentration-dependently cytotoxic effects against the three cancerous cell lines, fibroblast cells showed somehow more compatibility with chitosan. On the other hand, there were no significant differences between LMWC and HMWC cytotoxicity in all cell lines. The flow cytometry results showed the apoptosis pattern of death more in Saos-2 and HeLa while necrosis was more observable with MCF7. Also higher viability with both types of chitosan was seen in fibroblast as normal cells Conclusion: Chitosan shows anticancerous effect against 3 cancerous cell lines, while it is compatible with normal diploid fibroblast cells. Furthermore, it seems that the molecular weight of chitosan does not affect its anticancerous property.

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Combined Application of Pan-AKT Inhibitor MK-2206 and BCL-2 Antagonist Venetoclax in B-Cell Precursor Acute Lymphoblastic Leukemia

International Journal of Molecular Sciences ◽

10.3390/ijms22052771 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2771

Author(s):

Anna Richter ◽

Elisabeth Fischer ◽

Clemens Holz ◽

Julia Schulze ◽

Sandra Lange ◽

...

Keyword(s):

Cell Proliferation ◽

Tumor Cell ◽

Cell Lines ◽

Morphological Changes ◽

Synergistic Effects ◽

Lymphoblastic Leukemia ◽

Tumor Cell Proliferation ◽

Akt Inhibitor ◽

Akt Phosphorylation ◽

Combined Application

Aberrant PI3K/AKT signaling is a hallmark of acute B-lymphoblastic leukemia (B-ALL) resulting in increased tumor cell proliferation and apoptosis deficiency. While previous AKT inhibitors struggled with selectivity, MK-2206 promises meticulous pan-AKT targeting with proven anti-tumor activity. We herein, characterize the effect of MK-2206 on B-ALL cell lines and primary samples and investigate potential synergistic effects with BCL-2 inhibitor venetoclax to overcome limitations in apoptosis induction. MK-2206 incubation reduced AKT phosphorylation and influenced downstream signaling activity. Interestingly, after MK-2206 mono application tumor cell proliferation and metabolic activity were diminished significantly independently of basal AKT phosphorylation. Morphological changes but no induction of apoptosis was detected in the observed cell lines. In contrast, primary samples cultivated in a protective microenvironment showed a decrease in vital cells. Combined MK-2206 and venetoclax incubation resulted in partially synergistic anti-proliferative effects independently of application sequence in SEM and RS4;11 cell lines. Venetoclax-mediated apoptosis was not intensified by addition of MK-2206. Functional assessment of BCL-2 inhibition via Bax translocation assay revealed slightly increased pro-apoptotic signaling after combined MK-2206 and venetoclax incubation. In summary, we demonstrate that the pan-AKT inhibitor MK-2206 potently blocks B-ALL cell proliferation and for the first time characterize the synergistic effect of combined MK-2206 and venetoclax treatment in B-ALL.

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Ruxolitinib Combined with Gemcitabine against Cholangiocarcinoma Growth via the JAK2/STAT1/3/ALDH1A3 Pathway

Biomedicines ◽

10.3390/biomedicines9080885 ◽

2021 ◽

Vol 9 (8) ◽

pp. 885

Author(s):

Shin-Yi Chung ◽

Yi-Ping Hung ◽

Yi-Ru Pan ◽

Yu-Chan Chang ◽

Chiao-En Wu ◽

...

Keyword(s):

Cell Lines ◽

Nuclear Translocation ◽

Vital Role ◽

First Line ◽

Current Standard ◽

Integrated Network ◽

Jak2 Inhibitor ◽

Therapeutic Drugs ◽

Cholangiocarcinoma Cell ◽

Malignant Behavior

Cholangiocarcinoma is the most common primary malignant tumor of the bile duct. The current standard first-line treatment for advanced or metastatic cholangiocarcinoma is gemcitabine and cisplatin. However, few effective treatment choices exist for refractory cholangiocarcinoma, and additional therapeutic drugs are urgently required. Our previous work demonstrated that the ALDH isoform 1A3 plays a vital role in the malignant behavior of cholangiocarcinoma and may serve as a new therapeutic target. In this study, we found a positive correlation between ALDH1A3 protein expression levels and the cell migration abilities of three cholangiocarcinoma cell lines, which was verified using ALDH1A3-overexpressing and ALDH1A3-knockdown clones. We also used ALDH1A3-high and ALDH1A3-low populations of cholangiocarcinoma cell lines from the library of integrated network-based cellular signatures (LINCS) program and assessed the effects of ruxolitinib, a commercially available JAK2 inhibitor. Ruxolitinib had a higher cytotoxic effect when combined with gemcitabine. Furthermore, the nuclear translocation STAT1 and STAT3 heterodimers were markedly diminished by ruxolitinib treatment, possibly resulting in decreased ALDH1A3 activation. Notably, ruxolitinib alone or combined with gemcitabine led to significantly reduced tumor size and weight. Collectively, our studies suggest that ruxolitinib might suppress the ALDH1A3 activation through the JAK2/STAT1/3 pathway in cholangiocarcinoma, and trials should be undertaken to evaluate its efficacy in clinical therapy.

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Combined Inhibition of AKT and KIT Restores Expression of Programmed Cell Death 4 (PDCD4) in Gastrointestinal Stromal Tumor

Cancers ◽

10.3390/cancers13153699 ◽

2021 ◽

Vol 13 (15) ◽

pp. 3699

Author(s):

Marya Kozinova ◽

Shalina Joshi ◽

Shuai Ye ◽

Martin G. Belinsky ◽

Dinara Sharipova ◽

...

Keyword(s):

Cell Death ◽

Gastrointestinal Stromal Tumor ◽

Cell Lines ◽

Synergistic Effects ◽

Single Agent ◽

Xenograft Model ◽

Stromal Tumor ◽

In Vivo Studies ◽

Preclinical Model ◽

Kit Mutation

The majority of gastrointestinal stromal tumor (GIST) patients develop resistance to the first-line KIT inhibitor, imatinib mesylate (IM), through acquisition of secondary mutations in KIT or bypass signaling pathway activation. In addition to KIT, AKT is a relevant target for inhibition, since the PI3K/AKT pathway is crucial for IM-resistant GIST survival. We evaluated the activity of a novel pan-AKT inhibitor, MK-4440 (formerly ARQ 751), as monotherapy and in combination with IM in GIST cell lines and preclinical models with varying IM sensitivities. Dual inhibition of KIT and AKT demonstrated synergistic effects in IM-sensitive and -resistant GIST cell lines. Proteomic analyses revealed upregulation of the tumor suppressor, PDCD4, in combination treated cells. Enhanced PDCD4 expression correlated to increased cell death. In vivo studies revealed superior efficacy of MK-4440/IM combination in an IM-sensitive preclinical model of GIST compared with either single agent. The combination demonstrated limited efficacy in two IM-resistant models, including a GIST patient-derived xenograft model possessing an exon 9 KIT mutation. These studies provide strong rationale for further use of AKT inhibition in combination with IM in primary GIST; however, alternative agents will need to be tested in combination with AKT inhibition in the resistant setting.

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