Increase In sub-G1 Population Research Articles

Okanin Inhibits Cell Growth and Induces Apoptosis and Pyroptosis in Oral Cancer.

Okanin, a flavonoid compound derived from Bidens pilosa L., has garnered attention for its anti-inflammatory properties. Although Bidens pilosa is commonly used in healthcare products and functional foods, the anticancer potential of okanin, particularly in oral cancer, remains underexplored. This study aims to investigate the effects of okanin on oral cancer cell lines and its potential as a therapeutic agent. The study involved assessing the cytotoxic effects of okanin on oral cancer cell lines SAS, SCC25, HSC3, and OEC-M1. The IC50 values were determined using methylene blue assays, and the clonogenic capacity was evaluated through colony formation assays. Flow cytometry was used to analyze cell cycle progression and apoptosis. Caspase-3/7 activity assays and annexin V/7-AAD staining confirmed the induction of apoptosis and pyroptosis. In vivo efficacy was assessed using a SAS xenograft model, and immunohistochemical analysis of xenograft tissue was performed to examine pyroptosis-related markers. Okanin exhibited potent cytotoxic effects with IC50 values of 12.0 ± 0.8, 58.9 ± 18.7, 18.1 ± 5.3, and 43.2 ± 6.2 μM in SAS, SCC25, HSC3, and OEC-M1 cells, respectively. It caused dose- and time-dependent reductions in cell viability and significantly impaired clonogenic capacity. Flow cytometry revealed G2/M cell cycle arrest and increased sub-G1 population, indicating cell cycle disruption and death. Okanin induced both apoptosis and pyroptosis, as confirmed by caspase-3/7 activity and annexin V/7-AAD staining. In vivo, okanin reduced tumor growth and involved pyroptosis-related markers such as CASP1, GSDMC, GSDMD, and GSDME. Okanin demonstrates significant anticancer potential, particularly in oral cancer, by inducing both apoptosis and pyroptosis. Its efficacy in reducing tumor growth in vivo further supports its potential as a novel therapeutic option. Further mechanistic studies are needed to elucidate the pathways involved in okanin-mediated cell death and to explore its clinical applications.

Abstract B070: Antitumor effect of AZD5305, a selective PARP1 inhibitor, in breast and gastric cancer cells

Abstract Background: Poly (ADP-ribose) polymerase (PARP) 1 has multiple cellular functions including its crucial role in DNA damage response. PARP inhibitor (PARPi) blocks the synthesis of PAR polymers and traps PARP1 on the damage sites. Currently approved PARPis target both PARP1 and PARP2, but the inhibition of PARP2 can cause undesirable hematological toxicity. AZD5305 is a novel selective PARP1 inhibitor that can reduce the hematological toxicity by off-targeting of PARP2. We evaluated the anti-tumor effects and mechanism of action of AZD5305 in breast and gastric cancer cells in vitro. Methods: To investigate the anti-tumor effects of AZD5305, we used 2 cell lines of breast cancer cells (HCC1395 and MCF7) and 3 cell lines of human gastric cancer cells (SNU-601, SNU-668 and KATO Ⅲ). The cytotoxicity of AZD5305 was evaluated by colony formation assay (CFA) for 14 days with increasing concentration of AZD5305 (doses range: 0-5nM). Cell cycle was analyzed by flow cytometry and apoptotic cell death was assessed by annexin-Ⅴ/PI staining. DNA damage was detected by comet assay and cell immunofluorescence assay. Results: BRCA1/2 mutated HCC1395 and RAD51C deficient SNU-601 were sensitive to AZD5305 (IC50: 0.25 nM), and SNU-668 was moderately sensitive (IC50: 2.05nM), while MCF7 and KATO III were less-sensitive to AZD5305 (IC50 > 5 nM). AZD5305 induced the G2/M cell cycle arrest in a dose-dependent manner in HCC1395 and SNU-601, but not in MCF7 and KATO Ⅲ. Moreover, AZD5305 increased sub-G1 population in HCC1395 and SNU-601 cells indicating apoptotic cell death, which was also confirmed by annexin-V assay and the cleaved PARP and caspase-7. Phosphorylation of RPA, γH2AX, and the expression of molecules related to DNA damage response were increased in the sensitive cells. Moreover, comet tails which indicate fragmented DNA were observed in the sensitive cells, but not in insensitive cells. Conclusions: AZD5305, a novel PARP1 selective inhibitor, shows cytotoxic effect in HR defective breast and gastric cancer cells in vitro. AZD5305 accumulates DNA damage which leads to apoptotic cell death in sensitive cell lines, but not in less-sensitive cell lines. Citation Format: Sujin Ham, So Hyeon Kim, Hae Min Hwang, Minyoung Lee, Youlim Noh, Changyun Lee, Yu-Jin Kim, Jinyong Kim, Dae-Won Lee, Kyung-Hun Lee, Seock-Ah Im. Antitumor effect of AZD5305, a selective PARP1 inhibitor, in breast and gastric cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B070.

P10.05.A INDUCED SENESCENCE AND CALCIFICATION TO ANAPLASTIC MENINGIOMA

Abstract BACKGROUND Although the majority of meningiomas are slow-growing and benign, atypical and anaplastic meningiomas behave aggressively with a penchant for recurrence. The 5-year risk of progression for incompletely resected meningiomas is over 80 percents. Specifically, the 5-year survival rates are 78-91% and 41-65% for atypical and anaplastic meningiomas, respectively. On the other hand, it has been reported that benign meningiomas with psammoma bodies or those with bone hyperplasia at the attachment site and subtype of high calcification have a low growth rate. Here we show that BMP signals essential for bone formation, especially GREM2 upstream inhibitor of BMP signals, are involved in meningioma growth, senescence and calcification. MATERIAL AND METHODS 1) A long-term primary cultured meningioma and commercially available anaplastic meningioma (IOMM-lee and HKB-MM) are used. For immunohistochemistry and expression analysis, 87 surgical samples operated in our hospital were used.2) We observed changes in phenotype, proliferation rate and cell cycle by inhibiting BMP signal. Overexpression of GREM2, an upstream inhibitor of BMP signals, was performed in anaplastic meningiomas and analyzed comprehensively. Downloaded RNA-seq data from GREIN, a public database, and performed a comprehensive analysis of the extent of GREM2 expression and gene expression related to cellular senescence. RESULTS 1) Inhibition of BMP signaling reduced proliferation. Moreover, long term inhibition of BMP signals induced calcification. 2) Expression of GREM2 related to proliferation in surgical samples. GREM2 mainly localized surrounding of psammoma bodies. 3)Overexpression of GREM2 increased sub-G1 population and inhibits cell proliferation. 4)Expression of GREM2 shifts tryptophan catabolism from Kynurenine/NAD/NADH to serotonin production. 5) Comprehensive analysis using RNA-seq data from GREIN showed that high expression of GREM2 decreases oxidative phosphorylation. 6)Senescence assay showed that inhibition of BMP signaling induced senescence. CONCLUSION Inhibition of BMP signaling reduced proliferation and induced calcification. The underlying mechanism is shifting tryptophan catabolism from Kynurenine/NADH/NAD to serotonin production.

Efficacy of Eribulin Plus Gemcitabine Combination in L-Sarcomas.

Although the overall survival of advanced soft-tissue sarcoma (STS) patients has increased in recent years, the median progression-free survival is lower than 5 months, meaning that there is an unmet need in this population. Among second-line treatments for advanced STS, eribulin is an anti-microtubule agent that has been approved for liposarcoma. Here, we tested the combination of eribulin with gemcitabine in preclinical models of L-sarcoma. The effect in cell viability was measured by MTS and clonogenic assay. Cell cycle profiling was studied by flow cytometry, while apoptosis was measured by flow cytometry and Western blotting. The activity of eribulin plus gemcitabine was evaluated in in vivo patient-derived xenograft (PDX) models. In L-sarcoma cell lines, eribulin plus gemcitabine showed to be synergistic, increasing the number of hypodiploid events (increased subG1 population) and the accumulation of DNA damage. In in vivo PDX models of L-sarcomas, eribulin combined with gemcitabine was a viable scheme, delaying tumour growth after one cycle of treatment, being more effective in leiomyosarcoma. The combination of eribulin and gemcitabine was synergistic in L-sarcoma cultures and it showed to be active in in vivo studies. This combination deserves further exploration in the clinical context.

SH-PRO extract alleviates benign prostatic hyperplasia via ROS-mediated activation of PARP/caspase 3 and inhibition of FOXO3a/AR/PSA signaling in vitro and in vivo.

To target benign prostatic hyperplasia (BPH) as a common urinary disease in old men, in the current study, the antiproliferative and apoptotic mechanism of SH-PRO, a mixture of Angelica gigas and Astragalus membranaceus (2:1), was evaluated in BPH-1 cells and rats with testosterone-induced BPH. Herein, SH-PRO significantly reduced the viability of BPH-1 cells and dihydrotestosterone (DHT)-treated RWPE-1 cells. Also, SH-PRO increased the sub-G1 population in BPH-1 cells and consistently attenuated the expression of pro-PARP, pro-caspase 3, Bcl2, FOXO3a, androgen receptor (AR), and prostate-specific antigen (PSA) in BPH-1 cells and DHT-treated RWPE-1 cells. Of note, SH-PRO generated reactive oxygen species (ROS) in BPH-1 cells, while ROS inhibitor N-acetyl-l-cysteine (NAC) disturbed the ability of SH-PRO to reduce the expression of pro-PARP, FOXO3a, catalase, SOD, and increase sub-G1 population in BPH-1 cells. Furthermore, oral treatment of SH-PRO significantly abrogated the weight of the prostate in testosterone-treated rats compared to BPH control with the reduced expression of AR, PSA, and DHT and lower plasma levels of DTH, bFGF, and EGF with no toxicity. Overall, these findings highlight the antiproliferative and apoptotic potential of SH-PRO via ROS-mediated activation of PARP and caspase 3 and inhibition of FOXO3a/AR/PSA signaling as a potent anti-BPH candidate.

Metformin Enhances the Anti-Cancer Efficacy of Sorafenib via Suppressing MAPK/ERK/Stat3 Axis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) incidence, as well as related mortality, has been steadily increasing in the USA and across the globe, partly due to the lack of effective therapeutic options for advanced HCC. Though sorafenib is considered standard-of-care for advanced HCC, it only improves median survival by a few months when compared to placebo. Sorafenib is also associated with several unpleasant side effects that often lead to early abatement of therapy. Here, we investigate whether a combination regimen including low-dose sorafenib and a non-toxic dose of anti-diabetic drug metformin can achieve effective inhibition of HCC. Indeed, combining metformin with low-dose sorafenib inhibited growth, proliferation, migration, and invasion potential of HCC cells. We observed a 5.3- and 1.9-fold increase in sub-G1 population in the combination treatment compared to sorafenib alone. We found that the combination of metformin enhanced the efficacy of sorafenib and inhibited the MAPK/ERK/Stat3 axis. Our in vivo studies corroborated the in vitro findings, and mice harboring HepG2-derived tumors showed effective tumor reduction upon treatment with low-dose sorafenib and metformin combination. This work sheds light on a therapeutic strategy aiming to augment sorafenib efficacy or dose-de-escalation that may prove beneficial in circumventing sorafenib resistance as well as minimizing related side effects.

Biosynthesized CdS Nanoparticle Induces ROS-dependent Apoptosis in Human Lung Cancer Cells.

The World Health Organization (WHO) estimated that the number of cancer-related deaths was 9.6 million in 2018 and 2.09 million deaths occurred by lung cancer. The American Institute for Cancer Research (AICR) also observed gender preferences in lung cancer, common in men than women. Since the past decade, nanoparticles have now been widely documented for their anti-cancer properties, which signifies that the development of nanotechnology would be a future diagnosis and treatment strategy for lung cancer. The current study aimed to investigate the role of biosynthesized CdS nanoparticles (CdS NPs) in lung cancer cells (A549). Therefore, whether the CdS NP induces lung cancer cell death and the underlying mechanism is yet to be elucidated. Literature was searched from various archives of biomedical and life science journals. Then, CdS NPs were biosynthesized and characterized by traditional and cutting-edge protocols. The CdS NP-mediated cell death was elucidated following standard protocols. CdS NPs induced cytotoxicity towards A549 cells in a dose-dependent manner. However, such a death mechanism does not go through necrosis. Intracellular reactive oxygen species (ROS) accumulation and mitochondrial membrane depolarization demonstrated that cell death is associated with intracellular ROS production. Furthermore, increased sub-G1 population, Bax expression, and decreased Bcl-2 expression revealed that the death was caused by apoptosis. CdS NPs promote apoptosis-mediated lung cancer cell death through ROS production.

Abstract 5456: Discovery of OTS447, a highly potent and selective inhibitor of FLT3 for the treatment of AML patients with FLT3-ITD/TKD mutations

Abstract FMS-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in acute myeloid leukemia (AML), and its activating mutations including internal tandem duplications (ITDs) and missense point mutations in the tyrosine kinase domain (TKD) are found in approximately 30% of AML patients. Although the patients initially well responded to FLT3 inhibitors such as gilteritinib, the most cases relapsed within a few months after the initiation of treatment. For these patients who relapsed after FLT3 inhibitor-based therapy, no effective treatment is available. Mechanisms of resistance are not fully elucidated, but FLT3-ITD-TKD double mutant is considered as one of possible mechanisms. We have discovered a novel FLT3 inhibitor OTS447 that shows the potent and selective inhibition in both FLT3-ITD and FLT3-ITD-TKD double mutants. OTS447 was identified through the screening of our in-house proprietary compound library. It showed potent inhibitory activity against FLT3 with an IC50 value of 0.19 nM. The selectivity of OTS447 was investigated by human kinase profile assay. Among 371 human kinases tested, there were only seven (including FLT3) whose activity were inhibited by 80% or more at 5 nM of OTS447. We then examined the anti-proliferative effects in FLT3-ITD and -wild type (WT) AML cell lines. The proliferation of FLT3-ITD cell lines, MV4-11 and MOLM13, was more strongly suppressed than that of FLT3-WT cell lines. We also investigated the inhibitory effects to FLT3-ITD-TKD double mutants using Ba/F3 cells. FLT3-ITD-D835Y mutant was inhibited as strongly as FLT3-ITD mutant, and FLT3-ITD-F691I mutant was more strongly inhibited than Ba/F3 parental cells. These data suggest that OTS447 has selective inhibitory activity against FLT3 mutants. To confirm that FLT3 inhibition by OTS447 leads to the anti-proliferative effect, FLT3 autophosphorylation and phosphorylation of downstream molecules such as STAT5, ERK and AKT were examined. Both autophosphorylation of FLT3 and phosphorylation of downstream molecules were decreased by OTS447 in dose-dependent manner. Moreover, treatment of OTS447 induced the apoptosis and increased sub-G1 population in MV4-11 cell. Finally, we tested OTS447 anti-tumor effect using MV4-11 mouse xenograft model. OTS447 showed potent growth inhibition against MV4-11 tumor in dose-dependent manner. In summary, we discovered OTS447, a potent and selective FLT3 inhibitor, that can inhibit not only ITD mutation but also ITD-TKD mutations of the FLT3 gene. OTS447 possesses cytotoxic activity induced by inhibition of FLT3 signaling pathway and has anti-tumor activity in mouse xenograft model. We are pursuing further optimization of the inhibitor aiming for the effective treatment of AML patients with several types of FLT3-activating mutations. Citation Format: Yasuhide Okamoto, Naofumi Takamatsu, Takashi Miyamoto, Akiko Taira, Kozue Toyota, Yukiho Imai, Shinobu Fujii, Rumiko Ono, Kyoko Adachi, Yo Matsuo, Suyoun Chung, Jae-Hyun Park. Discovery of OTS447, a highly potent and selective inhibitor of FLT3 for the treatment of AML patients with FLT3-ITD/TKD mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5456.

Abstract 3023: Fatty acid synthase (FASN) inhibitors synergize with carfilzomib (CFZ) in acute myeloid leukemia (AML) and multiple myeloma (MM)

Abstract Background: Fatty acid synthase (FASN) upregulation during conditions of oxidative stress contributes to tumor proliferation and survival, which appears to be a mechanism of proteasome inhibitor resistance. Here we demonstrate that acute myeloid leukemia (AML) cell lines resistant to carfilzomib (CFZ), a second-generation proteasome inhibitor, have higher basal FASN expression and targeting FASN with small molecule inhibitors enhances the cytotoxic effect of CFZ in both AML and multiple myeloma (MM) cell lines. Methods: In a proliferation assay, human AML and MM cell lines were treated with a single dose of CFZ for 7 days. Inhibition of proliferation was defined using an IC50 cutoff for CFZ of 10nM for AML and 5nM for MM as the threshold for sensitivity. Sensitive and resistant cell lines were subjected to apoptosis and cell cycle analyses by flow cytometry after being exposed to CFZ for 72 hours. Proteomic analysis was performed at baseline using reverse phase protein assay (RPPA). For CFZ and FASN inhibitor combination assays, AML and MM cell lines with varying sensitivities to CFZ were exposed to CFZ and FASN inhibitors, orlistat or TVB-3166, simultaneously, and the apoptosis rate were analyzed by flow cytometry. For western blots, selected AML and MM cell lines were incubated with compounds for 24 hours, and the lysates were probed for selected targets. Results: Single-agent CFZ induced apoptosis in sensitive AML and MM cell lines, while apoptotic rates remained low in resistant cell lines. Cell cycle analysis showed increased sub-G1 population in sensitive cell lines compared to resistant cell lines. RPPA revealed that FASN, a key enzyme involved in lipogenesis, correlated with CFZ sensitivity, and CFZ resistant lines trended towards higher basal FASN levels. When CFZ was combined with FASN inhibitors, orlistat or TVB-3166, significant synergy was observed in the apoptosis assays in the AML and MM cell lines. Western blot analyses showed FASN inhibitors enhanced the anti-proliferation and pro-apoptotic effects of CFZ. Conclusion: CFZ demonstrated single agent activity in nanomolar ranges in human AML and MM cell lines. When combined with agents targeting lipid-metabolism, CFZ showed synergistic effect in apoptosis, suggesting this combination could potentially be a new therapeutic strategy for AML and MM. Citation Format: Maoyu Peng, Sanaz Noelle Ghafouri, Martina SJ McDermott, Dennis J. Slamon, Sarah M. Larson. Fatty acid synthase (FASN) inhibitors synergize with carfilzomib (CFZ) in acute myeloid leukemia (AML) and multiple myeloma (MM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3023.

Anticancer Activity of Cannabidiol (CBD) in Human Colorectal Cancer Cells: A Mechanistic Study

ObjectivesCannabidiol (CBD) is a major non-psychoactive bioactive component of the phytocannabinoids abundant in cannabis. Its potential use for treatment of colorectal cancer has been proposed by several studies. This study investigated the anti-cancer activity of CBD and the mechanism of that activity in human colorectal cancer cells. MethodsHuman colorectal cancer cell lines (SW620, SW480, HCT116, and Caco-2) and a human normal colon cell line (CCD18CO) were maintained in DMEM supplemented with 10% FBS and antibiotics. Cell viability was measured by MTT assay. Cell cycle phase was measured by flow cytometry. Apoptosis was analyzed by measuring caspase 3/7 enzyme activity. Western blotting was performed to compare expression of proteins regulating cell cycle arrest, apoptosis, and endoplasmic reticulum (ER) stress. Selective inhibitors for cannabinoid receptors were used to test the relevance of each receptor for CBD-mediated cell viability repression and apoptosis induction. Different types of CBD derivatives were tested for effects on cell viability. Data were analyzed using one-way analysis of variance (ANOVA), or two-way ANOVA followed by Bonferroni post-hoc tests (Dunnett T3 tests for unequal variance) for intergroup comparisons. ResultsCBD repressed viability of SW620, SW480, HCT116, and Caco-2 human colorectal cancer cells, with respective IC50 values of 5.4, 10.4, 10.8, and 20 μM for 24-hour treatment and 4.7, 5.8, 5.7, and 13.8 μM for 48-hour treatment. Moreover, CBD treatment led to G1-phase cell cycle arrest and an increased sub-G1 population with downregulation of cyclin D1, cyclin D3, CDK2, CDK4, and CDK6. It also increased activity of caspase 3/7, production of cleaved PARP, and expression of ER stress proteins (BiP, IRE1α, p-eIF2, ATF3 and ATF4). Repression of cell viability and induction of apoptotic cell death occurred through a mechanism dependent on cannabinoid receptor type 2 (CB2). Suppression of cell viability was also observed in cells treated with other of non-psychoactive cannabinoid derivatives (cannabidivarin, cannabigerol, cannabicyclol, cannabigerovarin). ConclusionsOur data indicate that CBD and its derivatives could be promising agents for the prevention of human colorectal cancer. Funding SourcesNo funding source.

Cannabidiol exerts anti-proliferative activity via a cannabinoid receptor 2-dependent mechanism in human colorectal cancer cells

Colorectal cancer is the third leading cause of cancer incidence and mortality in the United States. Cannabidiol (CBD), the second most abundant phytocannabinoid in Cannabis sativa, has potential use in cancer treatment on the basis of many studies showing its anti-cancer activity in diverse types of cancer, including colon cancer. However, its mechanism of action is not yet fully understood. In the current study, we observed CBD to repress viability of different human colorectal cancer cells in a dose-dependent manner. CBD treatment led to G1-phase cell cycle arrest and an increased sub-G1 population (apoptotic cells); it also downregulated protein expression of cyclin D1, cyclin D3, cyclin-dependent kinase 2 (CDK2), CDK4, and CDK6. CBD further increased caspase 3/7 activity and cleaved poly(ADP-ribose) polymerase, and elevated expression of endoplasmic reticulum (ER) stress proteins including binding immunoglobulin protein (BiP), inositol-requiring enzyme 1α (IRE1α), phosphorylated eukaryotic initiation factor 2α (eIF2α), activating transcription factor 3 (ATF3), and ATF4. We found that CBD repressed cell viability and induced apoptotic cell death through a mechanism dependent on cannabinoid receptor type 2 (CB2), but not on CB1, transient receptor potential vanilloid, or peroxisome proliferator-activated receptor gamma. Anti-proliferative activity was also observed for other non-psychoactive cannabinoid derivatives including cannabidivarin (CBDV), cannabigerol (CBG), cannabicyclol (CBL), and cannabigerovarin (CBGV). Our data indicate that CBD and its derivatives could be promising agents for the prevention of human colorectal cancer.

Different Cytotoxic Effects of Vetiver Oil on Three Types of Cancer Cells, Mainly Targeting CNR2 on TNBC.

Objective:To investigate vetiver oil (VO) selectivity effects on several cancer cell types and identify the β-caryophyllene role and mechanisms to prevent cancer development. Methods:Cytotoxic effects of VO on three types of cancer cells (WiDr, 4T1, T47D) were determined using MTT assay. VO’s effects on the cell cycle and apoptosis were analyzed using flow cytometry. Intracellular Reactive Oxygen Species (ROS) of cells after treatment with VO was observed with DCFDA staining. Bioinformatics study and molecular docking were used to determine the molecular targets of VO. Results:VO contained various essential oils in which β-caryophyllene was the most abundant. 4T1 cells performed the lowest IC50 value. WiDr and 4T1 cells showed an arrest in the G2/M phase, while T47D showed an increase of sub G1 population after VO treatment. On the other hand, apoptosis was only observed in WiDr and T47D cells. ROS levels were increased significantly in WiDr and T47D cells but not in 4T1 cells. Cannabinoids CB2 receptor (CNR2) was highly expressed in 4T1 cells and commonly exhibited a low survival rate on Triple Negative Breast Cancer (TNBC) patients. CNR2 was the notable target of β-caryophyllene and performed agonistic interaction, which might have contributed to its cytotoxic activity against 4T1 cells.Conclusion:The molecular interaction of VO cannabinoid agonists and the CNR2 receptor was the underlying cause of VO cytotoxicity, which is a VO distinction on TNBC. Therefore, VO is better suited for use as an anti-cancer agent in TNBC cells.

MiR193a-5p Mediated ZNF746 and c-Myc Signaling Axis Is Critically Involved in Morusin Induced Apoptosis in Colorectal Cancer Cells.

Novel target therapy is on the spotlight for effective cancer therapy. Hence, in the present study, the underlying apoptotic mechanism of Morusin was explored in association with miR193a-5p mediated ZNF746/c-Myc signaling axis in colorectal cancer cells (CRCs). Herein, Morusin reduced the viability and the number of colonies in HCT116 and SW480 CRCs. Additionally, Morusin increased sub-G1 population, cleavages of poly (ADP-ribose) polymerase (PARP) and caspase-3 and inhibited the expression of zinc finger protein 746 (ZNF746) and c-Myc in HCT116 and SW480 cells. Conversely, overexpression of ZNF746 suppressed the ability of Morusin to abrogate the expression of c-Myc in HCT116 cells, as ZNF746 enhanced the stability of c-Myc via their direct binding through nuclear colocalization in HCT116 cells by immunofluorescence and immunoprecipitation. Notably, Morusin upregulated miR193a-5p as a tumor suppressor, while miR193a-5p inhibitor masked the ability of Morusin to reduce the expression of ZNF746, c-Myc, and pro-PARP in HCT116 cells. To our knowledge, these findings provide the novel insight on miR193a-5p mediated inhibition of ZNF746/c-Myc signaling in Morusin induced apoptosis in CRCs.

Preclinical evaluation of the ETS inhibitor TK216 against relapsed and refractory childhood leukemia.

10033 Background: Although survival rates have improved in the recent past, relapse and refractory disease remain a significant cause of death in children with leukemia. This calls for an urgent need for the development of novel therapies that could effectively treat leukemias in children. The E26 transformation specific (ETS) family of transcription factors regulate various normal cellular functions but are abnormally expressed in various cancers, including leukemia. TK216 is an ETS inhibitor, that has shown pre-clinical activity and clinical efficacy in solid tumors. In this study, we explore the feasibility of using TK216 as a therapeutic agent for the treatment of high risk refractory pediatric leukemia. Methods: A panel of pediatric leukemia derived cell lines and primary blast cells representing a spectrum of molecular abnormalities seen in pediatric leukemia were treated in vitro with TK216 to determine cytotoxicity. Normal lymphocytes were used as controls and cell viability was determined 72 hours post-treatment by Alamar blue assay. The induction of tumor cell apoptosis and target modulation were detected by Western blotting. Alterations in the cell cycle were assessed by FACS analysis with PI staining. Drug combination studies were carried out with established anti-leukemic agents to identify synergy for greater therapeutic efficiency. Results: TK216 decreased cell viability in leukemia cells compared to normal lymphocyte controls in a dose-dependent manner with variations in sensitivity noted with inherent molecular abnormalities. The IC50 values observed ranged from 0.22 µM for the most sensitive cell line, MV4-11 to 0.95 µM for least sensitive cell line, SUP-B15. Apoptosis induction upon TK216 treatment was confirmed by PARP cleavage and caspase 3 activation. Cell cycle analysis demonstrated increased sub-G1 population of cells after TK216 treatment. A strong correlation between sub-G1 population and sensitivity of the cell line towards TK216 (47% in MV4-11 vs 3.72% in SUP-B15) was observed. Screening of a panel of 200 FDA approved anti-cancer agents in drug combination studies identified potential agents for drug synergy. Significant drug synergy was noted with TK216 in combination with the epigenetic modifier 5-azacytidine and the Bcl-2 inhibitor, Venetoclax. [Combination Index for Venetoclax and TK216, mean = 0.65 for MV4-11 and 0.33 for SUP-B15]. Conclusions: Data from our study demonstrate that the ETS inhibitor TK216 induces apoptosis and cell cycle arrest in pediatric leukemia cells at physiologically relevant concentrations. Our combination studies identified distinct anti-cancer agents that could be used for developing effective drug combination regimens with TK216. Overall, our findings provide essential preclinical data for the consideration of TK216 in early phase clinical trials for the treatment of selected high-risk and refractory childhood leukemia.

Hydromethanolic root and aerial part extracts from Echium arenarium Guss suppress proliferation and induce apoptosis of multiple myeloma cells through mitochondrial pathway.

Echium arenarium Guss is a Mediterranean plant traditionally used in healing skin wound and it was reported exhibiting potent antioxidant, antibacterial, and antiparasitic activities. However, antitumoral activities of this plant have not yet been explored. Here we investigated for the first time, root (EARE) and aerial part (EAAPE) extracts of E. arenarium Guss to examine cytotoxicity and apoptosis activation pathway on U266 human multiple myeloma (MM) cell line. We demonstrated that EARE and EAAPE decreased U266 cell viability in a dose dependent manner. Based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, EARE was significantly two times more efficient (IC50 value 41 μg/ml) than EAAPE (IC50 value 82 μg/ml) considering 48 h of treatment. Furthermore, after 24 h of exposure to 100 μg/ml of EARE or EAAPE, cell cycle showed remarkable increase in sub-G1 population and a decrease of U266 cells proportion in G1 phase. In addition, EARE increased cell percentage in S phase. Moreover, analysis revealed that EAAPE or EARE induced apoptosis of U266 cells after 24 h of treatment. Interestingly, depolarization of mitochondrial membrane potential and activation of caspase 3/7 were demonstrated in treated U266 cells. Phytochemical analysis of E. arenarium extracts showed that EARE exhibited the highest content of total phenolic content. Interestingly, six phenolic compounds were identified. Myricitrin was the major compound in EARE, followed by luteolin 7-O-glucoside, resorcinol, polydatin, Trans-hydroxycinnamic acid, and hyperoside. These findings proved that an intrinsic mitochondria-mediated apoptosis pathway probably mediated the apoptotic effects of E. arenarium Guss extracts on U266 cells, and this will suggest several action plans to treat MM.

The natural flavones, acacetin and apigenin, induce Cdk-Cyclin mediated G2/M phase arrest and trigger ROS-mediated apoptosis in glioblastoma cells.

Brain and CNS-related cancers are rare; however, 0.3 million incidences and 0.24 million deaths in 2018demonstrates the unrelenting associated dangers. Glioblastoma is a brain cancer of star-shaped glial cells. It is almost universally fatal within 2 years of diagnosis despite maximal medical therapies. This study aims to evaluate the in-depth anticancer activity of acacetin and apigenin on glioblastoma cells (U87). In the present report, we have isolated two flavonoids, acacetin and apigenin; and studied the in-depth anticancer activity on U87 cells. Selective cytotoxicity of acacetin and apigenin was observed towards the U87 cells (IC50: 43.73± 1.19 and 48.18± 1.37μM, respectively). The flow cytometer-based result revealed the induction of G2/M phase arrest along with the increase in sub G1 population upon compound treatment. Annexin-V-FLUOS and DAPI staining also confirmed the apoptosis-inducing effects of compounds. Flow cytometer and confocal microscopy-based DCFH-DA staining showed ROS-inducing effect of the compounds. The up-regulation of p21 and down-regulation of Cyclin-A1, Cyclin-B1, and Cdk-1 revealed the G2/M phase arrest mechanism of acacetin and apigenin. Furthermore, western blotting result confirmed the activation of intrinsic pathway of apoptosis upon acacetin treatment and activation of both extrinsic and intrinsic pathways of apoptosis upon apigenin treatment through the regulation of Bax, t-Bid, caspase 8, caspase 9, caspase 3, and PARP. The obtained result showed a significant effect (P < 0.05) of acacetin and apigenin on U87 cells. Acacetin and apigenin-induced ROS is responsible for the induction of cell cycle arrest and activation of caspase-cascade pathways in U87 cells.

Evaluation of Metastasis Suppressor Genes Expression and In Vitro Anti-Cancer Effects of Zinc Oxide Nanoparticles in Human Breast Cancer Cell Lines MCF-7 and T47D.

Background:Metallic nanoparticles are useful materials to be applied in biomedical research. In this study, the possible apoptotic and anti-metastatic activity of Zinc Oxide Nanoparticles (ZnONPs) was assessed in breast cancer cells.Methods:First, in vitro cell viability was investigated by MTT assay in two human breast cancer cells (MCF-7 and T47D) and normal Human Embryonic Kidney (HEK293) cells at 37°C overnight. Apoptosis induced by ZnONPs was evaluated by annexin V/PI staining, cell cycle analysis and caspase assay in cancerous cells. Moreover, quantitative real-time PCR was employed for the detection of two metastasis suppressor genes (KAI-1 and NM23) expression in cancerous cells.Results:Data demonstrated that ZnONPs exert a dose-dependent inhibitory effect on the viability of T47D and MCF-7 cells, while no cytotoxic effect was observed on normal HEK293 cells. The mRNA expression levels of KAI-1 and non-metastatic protein (NM23) genes were up-regulated in ZnONP-exposed cancerous cells. ZnONPs were also found to enhance the apoptosis properties of cells by annexin V/PI staining, and caspase assay in cancerous cells. Furthermore, ZnONPs can increase sub-G1 population as compared to negative control.Conclusion:Our findings showed that ZnONPs induce apoptotic activity and can modulate metastasis by up-regulating of KAI-1 and NM23 gene expression in two breast cancer (MCF-7 and T47D) cells.

Potent anti-inflammatory Terminalia chebula fruit showed in vitro anticancer activity on lung and breast carcinoma cells through the regulation of Bax/Bcl-2 and caspase-cascade pathways.

The present study was aimed to investigate the anticancer and anti-inflammatory activities of Terminalia chebula fruit (TCME). The TCME was evaluated for in vitro anticancer activity on A549 and MCF-7 cells. TCME showed cytotoxicity toward A549 (IC50 - 359.06±20.04µg/ml), and MCF-7 (IC50 - 61.02±5.55µg/ml) cells. The flow-cytometer analysis revealed increase in sub G1 population and apoptotic population, which were observed through cell cycle analysis and annexin-V-FLUOS staining. Confocal microscopy showed DNA fragmentation in both the cell lines upon TCME treatment. Moreover, TCME treatment induces activation of apoptosis-related caspase-cascade pathways in both the cell lines. TCME treatment on RAW 264.7 cells revealed the anti-inflammatory properties by regulating nitrite and TNF-α production; iNOS, COX-2 levels, and translocation of NF-κB protein. Finally, HPLC analysis revealed the bioactive phytocompounds present in TCME. In conclusion, the combined results showed the potent anticancer and anti-inflammatory properties of T. chebula fruit. PRACTICAL APPLICATIONS: Lung cancer is a leading cause of death in men with 35.5% incidences and 30.8% mortality rate worldwide. On the contrary, breast cancer possesses 55.2% incidences and 16.6% mortality rate among the female worldwide. The present findings revealed the anti-lung and -breast cancer activity along with the potent anti-inflammatory potentials of Terminalia chebula fruit. These findings will helpful to isolate the active drug molecules from the Terminalia chebula fruit and mark them as an anticancer and anti-inflammatory agent.

Ribosomal protein L5 mediated inhibition of c-Myc is critically involved in sanggenon G induced apoptosis in non-small lung cancer cells.

Though Sanggenon G (SanG) from root bark of Morus alba was known to exhibit anti-oxidant and anti-depressant effects, its underlying mechanisms still remain unclear. Herein SanG reduced the viability of A549 and H1299 non-small lung cancer cells (NSCLCs). Also, SanG increased sub-G1 population via inhibition of cyclin D1, cyclin E, CDK2, CDK4 and Bcl-2, cleavages of poly (ADP-ribose) polymerase (PARP) and caspase-3 in A549 and H1299 cells. Of note, SanG effectively inhibited c-Myc expression by activating ribosomal protein L5 (RPL5) and reducing c-Myc stability even in the presence of cycloheximide and 20% serum in A549 cells. Furthermore, SanG enhanced the apoptotic effect with doxorubicin in A549 cells. Taken together, our results for the first time provide novel evidence that SanG suppresses proliferation and induces apoptosis via caspase-3 activation and RPL5 mediated inhibition of c-Myc with combinational potential with doxorubicin.

Novel synthetic aromatic thiourea derivatives and investigations on their cytotoxic potential efficacy

AbstractAromatic thiourea derivatives were synthesized by standard reactions from isothiocyanates in high yield. An efficient one‐pot synthesis of primary amine with carbon disulfide involving the intermediate dithiocarbamate conversion using T3P (propane phosphonic anhydride) to isothiocyanate is reported. Newly synthesized compounds showed excellent luminescence property. Cytotoxic investigation with different human cancer cell lines showed IC50 values varying from of 17 to 40 μM for N‐phenethyl‐3,4‐dihydroisoquinoline‐2(1H)‐carbothioamide (SCR172) among other molecules. Increase in Sub G1 population and increased depolarisation of mitochondria were evident in cells treated with SCR172, suggesting its role as a cancer therapeutic agent. This provides a lead structure for further synthetic modifications.