sub-G1 Cell Population Research Articles

Photoprotective Effect of Ultrasonic-Assisted Ethanol Extract from Sargassum horneri on UVB-Exposed HaCaT Keratinocytes

The present study investigated the photoprotective effect of the ultrasonic-assisted ethanol extract (USHE) from Sargassum horneri, a brown seaweed containing fucosterol (6.22 ± 0.06 mg/g), sulfoquinovosyl glycerolipids (C23H43O11S, C25H45O11S, C25H47O11S, C27H49O11S), and polyphenols, against oxidative damage in ultraviolet B (UVB)-exposed HaCaT keratinocytes. USHE indicated antioxidant activity in ferric-reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging. After screening experiments, 15.6, 31.3, and 62.5 µg/mL concentrations of USHE and ascorbic acid as positive control were selected to be used throughout the investigation. USHE increased cell viability by markedly reducing the production of intracellular reactive oxygen species (ROS) in UVB-exposed HaCaT keratinocytes. Additionally, USHE reduced the apoptosis and sub-G1 cell population and increased the mitochondrial membrane potential. Moreover, USHE modulated the protein expression levels of anti-apoptotic molecules (Bcl-xL, Bcl-2, and PARP) and pro-apoptotic molecules (Bax, cleaved caspase-3, p53, cleaved PARP, and cytochrome C). This modulation accorded with the upregulation of cytosolic heme oxygenase (HO)-1, NAD(P)H quinone oxidoreductase 1 (NQO 1), and nuclear factor erythroid-2-related factor 2 (Nrf2), collectively known as components of the antioxidant system. These findings suggest that USHE has a photoprotective effect on UVB-exposed HaCaT keratinocytes and can be utilized to develop cosmeceuticals for UVB protection.

Sodium butyrate activates the extrinsic and intrinsic apoptotic processes in murine cementoblasts

Background/purpose: The metabolic by-product butyric acid of Gram-negative anaerobic bacteria can invoke pathological effects on periodontal cells resulting in inflammation and further destruction of periodontium. However, limited researches on the effects of butyric acid on cementoblasts were reported. Therefore, this study aimed to investigate the type of cell death in murine cementoblast (OCCM.30) caused by adding the different concentrations of sodium butyrate to the cell culture. Materials and methodsOCCM.30 cells were exposed to sodium butyrate (0, 2, 4, 8, 16 mM) for 48 h. Cell viability was determined by microculture tetrazolium assay. Cell cycle distribution and cell death were analyzed by flow cytometry. Caspase-mediated apoptotic cascade was evaluated by Western blot. ResultsThe concentrations of sodium butyrate≧4 mM were found to inhibit cell viability of OCCM.30 cells in a dose-dependent manner (P < 0.05). Sodium butyrate elevated sub-G1 cell population which exhibited cell apoptosis in OCCM.30 cells (P < 0.05). In addition, early and later apoptotic cells were found in sodium butyrate-induced cell death. Sodium butyrate significantly stimulated the degradation of procaspases-3, -8, and -9 levels, respectively (P < 0.05). Simultaneously, sodium butyrate corresponded to augment the levels of cleaved forms of caspases-3, -8, and -9, respectively (P < 0.05). ConclusionTaken together, sodium butyrate is a cytotoxic agent and can induce apoptosis on cementoblasts. The pathway involved in apoptosis is activated by caspase family signaling pathways. These evidences may provide a new mechanistic insight into the mechanism of damage of cementoblasts during the development and progression of periodontitis.

Novel strategies in HPV‑16‑related cervical cancer treatment: An in vitro study of combined siRNA-E5 with oxaliplatin and ifosfamide chemotherapy

BackgroundCervical cancer, primarily caused by HPV infection, remains a global health concern. Current treatments face challenges including drug resistance and toxicity. This study investigates combining E5-siRNA with chemotherapy drugs, Oxaliplatin and Ifosfamide, to enhance treatment efficacy in HPV-16 positive cervical cancer cells, targeting E5 oncoprotein to overcome limitations of existing therapies. MethodsThe CaSki cervical cancer cell line was transfected with E5-siRNA, and subsequently treated with Oxaliplatin/Ifosfamide. Quantitative real-time PCR was employed to assess the expression of related genes including p53, MMP2, Nanog, and Caspases. Cell apoptosis, cell cycle progression, and cell viability were evaluated using Annexin V/PI staining, DAPI staining, and MTT test, respectively. Furthermore, stemness ability was determined through a colony formation assay, and cell motility was assessed by wound healing assay. ResultsE5-siRNA transfection significantly reduced E5 mRNA expression in CaSki cells compared to the control group. The MTT assay revealed that monotherapy with E5-siRNA, Oxaliplatin, or Ifosfamide had moderate effects on cell viability. However, combination therapy showed synergistic effects, reducing the IC50 of Oxaliplatin from 11.42 × 10−8 M (45.36 μg/ml) to 6.71 × 10−8 M (26.66 μg/ml) and Ifosfamide from 12.52 × 10−5 M (32.7 μg/ml) to 8.206 × 10−5 M (21.43 μg/ml). Flow cytometry analysis demonstrated a significant increase in apoptosis for combination treatments, with apoptosis rates rising from 11.02 % (Oxaliplatin alone) and 16.98 % (Ifosfamide alone) to 24.8 % (Oxaliplatin + E5-siRNA) and 34.9 % (Ifosfamide + E5-siRNA). The sub-G1 cell population increased from 15.7 % (Oxaliplatin alone) and 18 % (Ifosfamide alone) to 21.9 % (Oxaliplatin + E5-siRNA) and 27.1 % (Ifosfamide + E5-siRNA), indicating cell cycle arrest. The colony formation assay revealed a substantial decrease in the number of colonies following combination treatment. qRT-PCR analysis showed decreased expression of stemness-related genes CD44 and Nanog, and migration-related genes MMP2 and CXCL8 in the combination groups. Apoptosis-related genes Casp-3, Casp-9, and pP53 showed increased expression following combination therapy, while BAX expression increased and BCL2 expression decreased relative to the control. ConclusionThe study demonstrates that combining E5-siRNA with Oxaliplatin or Ifosfamide enhances the efficacy of chemotherapy in HPV-16 positive cervical cancer cells. This synergistic approach effectively targets multiple aspects of cancer cell behavior, including proliferation, apoptosis, migration, and stemness. The findings suggest that this combination strategy could potentially allow for lower chemotherapy doses, thereby reducing toxicity while maintaining therapeutic efficacy. This research provides valuable insights into targeting HPV E5 as a complementary approach to existing therapies focused on E6 and E7 oncoproteins, opening new avenues for combination therapies in cervical cancer treatment.

Targeting CK1δ and CK1ε as a New Therapeutic Approach for Clear Cell Renal Cell Carcinoma.

Kidney cancer ranks as the ninth most common cancer in men and the fourteenth in women globally, with renal cell carcinoma (RCC) being the most prevalent type. Despite advances in therapeutic strategies targeting angiogenesis and immune checkpoints, the absence of reliable markers for patient selection and limited duration of disease control underline the need for innovative approaches. CK1δ and CK1ε are highly conserved serine/threonine kinases involved in cell cycle regulation, apoptosis, and circadian rhythm. While CK1δ dysregulation is reportedly associated with breast and bladder cancer progression, their role in RCC remains elusive. This study aimed to investigate the feasibility of CK1δ/ε as new therapeutic targets for RCC patients. The relationship between CK1δ/ε and RCC progression was evaluated by the analysis of microarray dataset and TCGA database. The anticancer activity of CK1δ/ε inhibitor was examined by MTT/SRB assay, and apoptotic cell death was analyzed by flow cytometry and Western blotting. Our data demonstrate that the gene expression of CSNK1D and CSNK1E is significantly higher in clear cell RCC (ccRCC) tissues compared to normal kidney samples, which is correlated with lower survival rates in ccRCC patients. SR3029, a selective inhibitor targeting CK1δ/ε, significantly suppresses the viability and proliferation of ccRCC cell lines regardless of the status of VHL deficiency. Importantly, the inhibitor promotes the population of subG1 cells and induces apoptosis, and ectopically expression of CK1δ partially rescued SR3029-induced apoptosis in ccRCC cells. These findings underscore the crucial role of CK1δ and CK1ε in ccRCC progression, suggesting CK1δ/ε inhibitors as new therapeutic options for ccRCC patients.

The anticancer impact of folate-linked ZnO-decorated bovine serum albumin/silibinin nanoparticles on human pancreatic, breast, lung, and colon cancers

In the current study, we aimed to design an individual hybrid silibinin nano-delivery system consisting of ZnO and BSA components to study its antioxidant activity and apoptotic potential on human pancreatic, breast, lung, and colon cancer cell lines. The folate-linked ZnO-decorated bovine serum albumin/silibinin nanoparticles (FZBS-NP) were synthesized and characterized by FTIR, FESEM, DLS, and zeta potential analysis. The FZBS-NP’s cytotoxicity was evaluated by measuring the cancer cells’ (MCF-7, A549, HT-29, and Panc) viability. Moreover, the apoptotic potential of the nanoparticles was studied by conducting several analyses including AO/PI and DAPI cell staining analysis, apoptotic gene expression profile (BAX, BCL2, and Caspase-8) preparation, and FITC Annexin V/PI flow cytometry. Finally, both antioxidant assays (ABTS and DPPH) were utilized to analyze the FZBS-NPs’ antioxidant activities. The 152-nm FZBS-NP significantly induced the selective apoptotic death on the MCF-7, A549, HT-29, Panc, and Huvec cancer cells by increasing the SubG1 cell population following the increased treatment concentrations of FZBS-NP. Moreover, the FZBS-NPs exhibited powerful antioxidant activity. The BSA component of the FZBS-NPs delivery system improves the ability of the nanoparticles to gradually release silibinin and ZnO near the cancer cells. On the other hand, considering the powerful antioxidant activity of FZBS-NP, they have the potential to selectively induce apoptosis in human colon and breast cancer cells and protect normal types, which makes it an efficient safe anticancer compound. However, to verify the FZBS-NP anti-cancer efficiency further cancer and normal cell lines are required to measure several types of apoptotic gene expression.

A Novel 2-Methoxyestradiol Derivative: Disrupting Mitosis Inhibiting Cell Motility and Inducing Apoptosis in HeLa Cells In Vitro.

The clinical application of 2-methoxyestradiol (2ME) in cancer therapy has been limited by its low solubility and rapid metabolism. Derivatives of 2ME have been synthesised to enhance bioavailability and decrease hepatic metabolism. Compound 4a, an analog of 2ME, has demonstrated exceptional pharmacological activity, in addition to promising pharmacokinetic profile. Our study, therefore, aimed at exploring the anticancer effects of 4a on the cervical cancer cell line, HeLa. Compound 4a exhibited a significant and dose-dependent antimetastatic and antiinvasive impact on HeLa cells, as determined by wound-healing and Boyden chamber assays, respectively. Hoechst/Propidium iodide (HOPI) double staining showcased a substantial induction of apoptosis via 4a, with minimal necrotic effect. Flow cytometry revealed a significant G2/M phase arrest, accompanied by a noteworthy rise in the sub-G1 cell population, indicating apoptosis, 18 h post-treatment. Moreover, a cell-independent tubulin polymerisation assay illustrated compound 4a's ability to stabilise microtubules by promoting tubulin polymerisation. Molecular modelling experiments depicted that 4a interacts with the colchicine-binding site, nestled between the α and β tubulin dimers. Furthermore, 4a displayed an affinity for binding to and activating ER-α, as demonstrated by the luciferase reporter assay. These findings underscore the potential of 4a in inhibiting HPV18+ cervical cancer proliferation and cellular motility.

THE APOPTOSIS INDUCTION OF Zingiber oficinale ETHANOLIC EXTRACT-Treated HeLa (HUMAN CERVICAL CANCER) CELLS AND ACTIVE COMPOUND PROFILING USING GAS CHROMATOGRAPHY/MASS SPECTROMETRY

Cervical cancer accounts for the highest percentage of cancer-related deaths in Indonesia, accounting for nearly 60% of all cancer cases. Therefore, research into the anticancer mechanisms needs to be conducted. The ethanolic extract of Zingiber officinale (EEZO) contains zingiberene, a chemical known for its anticancer activity. Understanding the mechanism underlying the apoptosis-inducing effects is crucial. This study aimed to elucidate the apoptotic pathway and analyze the gas chromatography/mass spectrometry (GC/MS) profile of EEZO cells. The research commenced with the maceration of Zingiber officinale rhizomes using 75% ethanol to obtain EEZO. Apoptosis assays were conducted on both a negative control group and an EEZO-treated group of HeLa cells (cervical cancer cells). The apoptotic mechanism was evaluated using forward scattered light-side scattered light (FSC-SSC), fluorescein isothiocyanate (FTIC), and phycoerythrin (PE) flow cytometry. Apoptotic results were analyzed by comparing the control and EEZO samples, which revealed the number of viable cells, apoptotic cells, and cells in the sub-G1 phase. The major constituent of EEZO, which is expected to be a potent apoptosis inducer, was detected using GC/MS. The FSC-SSC results indicated a lower number of viable cells in the EEZO-exposed group than in the control group. FTIC results demonstrated that EEZO significantly increased apoptotic cell death, increasing from 68 to 1537 cells. PE flow cytometry revealed an elevated sub-G1 cell population, indicating the induction of apoptosis by EEZO. GC/MS analysis revealed five dominant components in EEZO, which had the potential to induce apoptosis: L-borneol, zingiberene, farnesol, beta-sesquiphellandrene, and alpha-curcumene ...

An Active Compound from the Pyrazine Family Induces Apoptosis by Targeting the Bax/Bcl2 and Survivin Expression in Chronic Myeloid Leukemia K562 Cells.

It has been established that pyrazine derivatives, which have widespread bioactivities, can effectively treat cancer. In this study, we investigated the effects of 2-methoxy-5-(oxiran-2-ylmethyl) phenyl pyrazine-2- carboxylate (2-mOPP), a new pyrazine derivative, on proliferation, viability, and apoptosis induction in human leukemia K562 cells. For this purpose, the K562 cells were treated with various concentrations (20-120 μM) of the 2-mOPP for 24-72 hours. Cell viability was determined by MTT growth inhibition assay. Apoptotic activity of 2-mOPP was investigated morphologically by Hoechst staining, cell surface expression assay of phosphatidylserine by Annexin-V/PI technique, as well as DNA fragmentation assay. The effect of 2-mOPP on the K562 cell cycle was studied by flow cytometry. To determine the impact of 2-mOPP on the expression of intrinsic apoptosis-related genes, Bcl2 (anti-apoptotic), Bax (pro-apoptotic), and Survivin genes expression levels were evaluated before and after treatment with 2-mOPP through Real-Time PCR analysis. The results revealed that 2-mOPP inhibited viability with IC50 of 25μM in 72 h. Morphological changes assessment by fluorescence microscopy, Annexin V/PI double staining by flow cytometry, and DNA ladders formation upon cell treatment with the 2-mOPP showed that this compound induces apoptosis at IC50 value. Cell cycle arrest was observed in the G0/G1 phase, and the sub-G1 cell population (the sign of apoptosis) increased in a time-dependent manner. Low expression levels of Bcl2 and Survivin in K562 cells were observed 24-72 h after treatment. Along with the down-regulation of Survivin and Bcl2, the expression of Bax was increased after treatment with 2-mOPP. These findings demonstrate that the new pyrazine derivative plays a crucial role in blocking the proliferation of the leukemic cells by inducing cell cycle arrest and apoptosis.

Minocycline declines interleukin-1ß-induced apoptosis and matrix metalloproteinase expression in C28/I2 chondrocyte cells: an in vitro study on osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease that occurs with aging. In its late phases, it is determined by the loss of chondrocytes and the breakdown of the extracellular matrix, resulting in pain and functional impairment. Interleukin-1 beta (IL-1β) is increased in the injured joints and contributes to the OA pathobiology by inducing chondrocyte apoptosis and up-regulation of matrix metalloproteinases (MMPs). Here, we aimed to understand whether minocycline could protect chondrocytes against the IL-1β-induced effects. The human C28/I2 chondrocyte cell line was treated with IL-1β or IL-1β plus minocycline. Cell viability/toxicity, cell cycle progression, and apoptosis were assessed with MMT assay and flow cytometry. Expression of apoptotic genes and MMPs were evaluated with qRT-PCR and western blotting. IL-1β showed a significant cytotoxic effect on the C28/I2 chondrocyte cells. The minocycline effective concentration (EC50) significantly protected the C28/I2 cells against the IL-1β-induced cytotoxic effect. Besides, minocycline effectively lowered IL-1β-induced sub-G1 cell population increase, indicating the minocycline anti-apoptotic effect. When assessed by real-time PCR and western blotting, the minocycline treatment group showed an elevated level of Bcl-2 and a significant decrease in the mRNA and protein expression of the apoptotic markers Bax and Caspase-3 and Matrix metalloproteinases (MMPs) such as MMP-3 and MMP-13. In conclusion, IL-1β promotes OA by inducing chondrocyte death and MMPs overexpression. Treatment with minocycline reduces these effects and decreases the production of apoptotic factors as well as the MMP-3 and MMP-13. Minocycline might be considered as an anti-IL-1β therapeutic supplement in the treatment of osteoarthritis. See also the graphical abstract(Fig. 1).

TRIB3 as a biomarker of gastric cancer cell sensitivity to chemotherapeutic agents running title: A protective role of TRIB3 on chemotherapy.

Understanding the role of TRIB3 in cellular chemotherapy responsiveness and survival could facilitate its development as a prognostic marker that could be used to improve chemotherapeutic efficiency against specific tumors. Therefore, the role of TRIB3 to reflect the cytotoxic abilities of chemotherapeutic agents was clarified in the tested gastric cancer cell lines. We have comprehensively investigated the protein expression of TRIB3 in three gastric cancer cell lines AGS, TMK-1, and MKN-45 cells treated with the anticancer drugs, 5-fluorouracil, cisplatin, and docetaxel. The Cell Count kit-8 was used to evaluate cell viability. Immunoblotting was performed to assay protein levels after drug treatment. Flow cytometry was carried out to evaluate the levels of sub-G1 cell population. Treatment of the tested gastric cancer cell lines dose-dependently decreased cell viability and protein levels of TRIB3 while increasing apoptosis. Overexpression of TRIB3 protects MKN-45 cells from endoplasmic reticulum stress-induced apoptosis but does not influence the induction of autophagy by anticancer drugs. In addition, overexpression of TRIB3 also rescued paroxetine-induced apoptosis and endoplasmic reticulum stress. Our previous and present results indicate that TRIB3 can protect gastric cancer cells against anticancer drug treatment and that downregulating TRIB3 may increase these cells' sensitivity to anticancer drugs. We thus suggest that the capability of anticancer drugs to downregulate TRIB3 can indicate tumors' potential susceptibility to these drugs.

Novel chemical scaffold as potential drug against Leishmania donovani: Integrated computational and experimental approaches.

In this study, we have screened a large number of Food and Drug Administration-approved compounds for novel anti-leishmanial molecules targeting the citrate synthase enzyme of the parasite. Based on their docking and molecular dynamic simulation statistics, five compounds were selected. These compounds followed Lipinski's rule of five. Additionally, in vitro, antileishmanial and cytotoxicity studies were performed. The three compounds, Abemaciclib, Bazedoxifene, and Vorapaxar, had shown effective anti-leishmanial activities with IC50 values of 0.92 ± 0.02, 0.65 ± 0.09, and 6.1 ± 0.91 against Leishmania donovani promastigote and with EC50 values of 1.52 ± 0.37, 2.11 ± 0.38, 10.4 ± 1.27 against intramacrophagic amastigote without significantly harming macrophage cells. Among them, from in silico and antileishmanial activities studies, Abemaciclib had been selected based on their less binding energy, good antileishmanial activities, and also a significant difference in their binding energy with human citrate synthase for cell death mechanistic studies using flow cytometry and a DNA fragmentation assay. The action of this compound resulted in an increased reactive oxygen species production, depolarization of mitochondrial membrane potential, DNA damage, and an increase in the sub-G1 cell population. These properties are the hallmarks of apoptosis which were further confirmed by apoptotic assay. Based on the above result, this anticancer compound Abemaciclib could be employed as a potential treatment option for leishmaniasis after further confirmation.

Naringenin Induces HepG2 Cell Apoptosis via ROS-Mediated JAK-2/STAT-3 Signaling Pathways.

Hepatocarcinoma is one of the most prevalent digestive system tumors worldwide and lacks effective therapy. Recently, naringenin has been isolated from some citrus fruits, and its anticancer effects have been tested. However, the molecular mechanisms of naringenin and the potential implications of oxidative stress in naringenin-induced cytotoxicity in HepG2 cells remain elusive. Based on the above, the present study examined the effect of naringenin on the cytotoxic and anticancer mechanisms of HepG2 cells. Naringenin-induced HepG2 cell apoptosis was confirmed via the accumulation of the sub-G1 cell population, phosphatidylserine exposure, mitochondrial transmembrane potential loss, DNA fragmentation, caspase-3 activation, and caspase-9 activation. Furthermore, naringenin enhanced cytotoxic effects on HepG2 cells and triggered intracellular reactive oxygen species; the signaling pathways of JAK-2/STAT-3 were inhibited, and caspase-3 was activated to advance cell apoptosis. These results suggest that naringenin plays an important role in inducing apoptosis in HepG2 cells and that naringenin may be a promising candidate for cancer therapy.

Abstract 950: Targeting PKMYT1 kinase is an effective treatment strategy in triple negative breast cancers with low molecular weight cyclin E (LMW-E) expression

Abstract Background: Cyclin E is post-translationally modified by neutrophil elastase mediated proteolytic cleavage to generate the low molecular weight isoforms of cyclin E (LMW-E) that are detected in various human cancers. We previously reported that 70% of triple negative breast cancers (TNBC) examined overexpress LMW-E, and these patients have a poor prognosis. Expression of LMW-E promotes genomic instability by causing DNA replication stress. PKMYT1 prevents premature mitotic entry by catalyzing CDK1 phosphorylation at T14, essential for preventing DNA damage and cell death when cyclin E, including LMW-E, is overexpressed. In this study, we tested the hypothesis that LMW-E positive status can be used as a biomarker of response in selecting TNBC patients who are likely to respond to RP-6306, a first in-class and selective inhibitor of PKMYT1 kinase. Results: Assessment of pre-treatment breast biopsies from TNBC patients (n=40) enrolled in a neoadjuvant chemotherapy prospective study for LMW-E and CDK1-pT14 revealed significant positive correlation between these two proteins. Furthermore, positivity of both biomarkers was associated with lack of pathologic complete response (pCR) to neoadjuvant chemotherapy. We next examined the mechanism of response to RP-6306 in vitro and in vivo using TNBC cell lines, patient-derived xenograft (PDX) models and transgenic mouse mammary tumor virus (MMTV) models expressing human LMW-E (hLMW-E). In vitro results using 7 different TNBC cell lines, revealed that high LMW-E levels are significantly predictive of response to RP-6306 (R2=0.78, p= 0.008), while LMW-E knockdown resulted in a 7X increase in IC50 values of RP-6306 (p&amp;lt;0.001). In high LMW-E cells, treatment with RP-6306 resulted in significant (i) downregulation of CDK1-pT14, PKMYT1, WEE1, cyclin B and pRb, (ii) accumulation of sub-G1 and polyploid cell population, (iii) apoptosis, (iv) accumulation of chromosomal breakage, (v) increased DNA damage (increase in γ-H2AX and 53BP1 foci/cell) and lack of DNA repair (downregulation of Rad51), and (vi) premature mitotic entry. Treatment of both breast cancer PDX models and hLMW-E transgenic tumors with RP-6306 revealed that only in animals with high LMW-E tumors, treatment results in significant decrease in tumor volume. However, RP-6306 was ineffective in reducing tumor volume in low cyclin E in vivo models. Immunohistochemical analysis revealed that in vivo efficacy of RP-6306 (in both PDX and transgenic models) was concomitant with increase in γ-H2AX and decrease in CDK1-pT14 and Ki67. Conclusion: Collectively, our results show that overexpression of LMW-E and CDK1-pT14 in TNBC can be used to stratify patients whose tumors are likely to respond to RP-6306. Mechanistically, LMW-E overexpressing TNBC cells activate CDK1 (in vitro and in vivo) to accelerate premature mitotic entry, leading to DNA damage and apoptosis. Citation Format: Amriti Lulla, Tuyen D. Nguyen, Mi Li, Sofia Mastoraki, Yan Wang, Tuyen Bui, Marc Pina, Spiridon Tsavachidis, Gary Marshall, Kelly K. Hunt, Khandan Keyomarsi. Targeting PKMYT1 kinase is an effective treatment strategy in triple negative breast cancers with low molecular weight cyclin E (LMW-E) expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 950.

UVC-Protective Activity of Lemongrass Among 12 Fat-soluble Herbal Extracts: Rapid Decay Due to Cytotoxicity.

The COVID-19 pandemic led to the rapid spread of the use of ultraviolet C (UVC) sterilizers in many public facilities. Considering the harmful effects of prolonged exposure to UVC, manufacturing of safe skin care products is an important countermeasure. In continuation of our recent study of water-soluble herbal extracts, the present study aimed at searching for anti-UVC components from fat-soluble herbal extracts. Human dermal fibroblast and melanoma cells were exposed to UVC (1.193 W/m2) for 3 min. Viable cell number was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell-cycle analysis was performed using a cell sorter. UVC-protective activity was quantified by the selective index (SI), i.e., the ratio of the 50% cytotoxic concentration for unirradiated cells to the concentration that restored viability of UVC-treated cells by 50%. Only lemongrass extract, among 12 fat-soluble herbal extracts, showed significant anti-UVC activity, comparable to that of lignified materials and tannins, but exceeding that of N-acetyl-L-cysteine and resveratrol. Lemongrass extract was highly cytotoxic, producing a subG1 cell population. During prolonged incubation in culture medium, the anti-UVC activity of lemongrass extract, sodium ascorbate and vanillic acid declined with an approximate half-life of <0.7, 5.4-21.6, and 27.8-87.0 h, respectively. Removal of cytotoxic principle(s) from lemongrass extract is crucial to producing long-lasting UVC-protective effects.

ADAMTS9-AS1 Long Non‑coding RNA Sponges miR‑128 and miR-150 to Regulate Ras/MAPK Signaling Pathway in Glioma.

Glioma is a malignancy of the central nervous system with a poor prognosis. Therefore, the elaboration of its molecular features creates therapeutic opportunities. Looking for the regulatory non-coding RNAs (lncRNAs and miRNAs) that are involved in glioma incidence/progression, RNA-seq analysis introduced upregulated ADAMTS9-AS1 as a bona fide candidate that sponges miR-128 and miR-150 and shows the negative correlation of expression with them. Then, RT-qPCR verified the upregulation of ADAMTS9-AS1 in glioma tissues and cell lines. Furthermore, dual-luciferase assay supported that cytoplasmic ADAMTS9-AS1 is capable of sponging miR-128 and miR-150, which are known as regulators of Ras/MAPK, PI3K, and Wnt pathways. Following the overexpression of ADAMTS9-AS1 in 1321N1 and U87 glioma cells, tyrosine kinase receptors (IGF1R and TrkC), as well as Wnt receptors (Lrp6 and Fzd) were upregulated, detected by RT-qPCR. Furthermore, downstream genes of both Ras/MAPK and Wnt pathways were upregulated. Finally following the ADAMTS9-AS1 overexpression, upregulation of Ras/MAPK and Wnt signaling pathways was verified through western blotting and Top/Fop flash assay, respectively. At the cellular level, ADAMTS9-AS1 overexpression brought about reduced sub-G1 cell population, increased proliferation rate, reduced apoptosis level, increased migration rate, shortened Bax/Bcl2 ratio, induced EMT, and stemness characteristics of transfected cells, detected by flow cytometry, MTT assay, scratch test, and RT-qPCR. Overall, these results introduced ADAMTS9-AS1 as an oncogene that upregulates Ras/MAPK and Wnt pathways through sponging of the miR-128 and miR-150 in glioma cells. The outcome of ADAMTS9-AS1 expression is more aggression of the glioma cells through increased EMT and stemness characteristics. These features candidate ADAMTS9-AS1 locus for glioma therapy. As a result, we discovered the oncogenic properties of ADAMTS9-AS1 in glioma cancer. It sponges miR-128 and miR-150 and subsequently overstimulates RAS/MAPK and Wnt signaling pathways, particularly at the receptors level. Thus, ADAMTS9-AS1 increases proliferation, migration, and stemness in glioma cell lines. A schematic representation showing the functional effect of ADAMTS9-AS1.

Bexarotene-induced cell death in ovarian cancer cells through Caspase-4-gasdermin E mediated pyroptosis

Bexarotene selectively activates retinoid X receptor, which is a commonly used anticancer agent for cutaneous T-cell lymphoma. In this study, we aimed to investigate the anticancer effect of bexarotene and its underlying mechanism in ovarian cancer in vitro. The ES2 and NIH:OVACAR3 ovarian cancer cell lines were treated with 0, 5, 10, or 20 µM of bexarotene. After 24 h, cell number measurement and lactate dehydrogenase (LDH) cytotoxicity assay were performed. The effect of bexarotene on CDKN1A expression, cell cycle-related protein, cell cycle, pyroptosis, and apoptosis was evaluated. Bexarotene reduced cell proliferation in all concentrations in both the cells. At concentrations of > 10 µM, extracellular LDH activity increased with cell rupture. Treatment using 10 µM of bexarotene increased CDKN1A mRNA levels, decreased cell cycle-related protein expression, and increased the sub-G1 cell population in both cells. In ES2 cells, caspase-4 and GSDME were activated, whereas caspase-3 was not, indicating that bexarotene-induced cell death might be pyroptosis. A clinical setting concentration of bexarotene induced cell death through caspase-4–mediated pyroptosis in ovarian cancer cell lines. Thus, bexarotene may serve as a novel therapeutic agent for ovarian cancer.

Effects of repeated exposure to 50 Hz electromagnetic field on breast cancer cells

ABSTRACT The extremely low frequency electromagnetic field (ELF-EMF) is emerging as a novel approach in cancer treatment. This study evaluated the impact of daily exposure to 50 Hz EMF on breast cancer cells in vitro. The MDA-MB-231 and MCF-7 cells were exposed to EMF (50 Hz 20 mT, for 3 hours per day for up to four days) and examined for cell vaibility. The effect of daily ELF-EMF exposure on cell cycle progression and cell death was further investigated. The result revealed that the consecutive exposure to 50 Hz EMF at 20 mT remarkably decreased the viability of MDA-MB-231 compared to the non-exposed group, while it had no significant effect on MCF-7 cells. The ELF-EMF exposure induced G1 phase arrest along with the increase in sub-G1 cell population in MDA-MB-231. Moreover, repeated exposure to 50 Hz EMF promoted cell cycle progression in MCF-7 by increasing the percentage of cells in the S phase. The fluorescent staining revealed that daily exposure of ELF-EMF induced apoptotic cell death in MDA-MB-231, but no morphological change was observed in MCF-7 cells. The results showed that repeated daily exposure to 50 Hz EMF exhibited anti-proliferative activity against invasive breast cancer cells by impairing cell cycle progression and inducing cell death.

Anticancer Effects of Propionic Acid Inducing Cell Death in Cervical Cancer Cells.

Recent studies found that short-chain fatty acids (SCFAs), which are produced through bacterial fermentation in the gastrointestinal tract, have oncoprotective effects against cervical cancer. The most common SCFAs that are well known include acetic acid, butyric acid, and propionic acid, among which propionic acid (PA) has been reported to induce apoptosis in HeLa cells. However, the mechanism in which SCFAs suppress HeLa cell viability remain poorly understood. Our study aims to provide a more detailed look into the mechanism of PA in HeLa cells. Flow cytometry analysis revealed that PA induces reactive oxygen species (ROS), leading to the dysfunction of the mitochondrial membrane. Moreover, PA inhibits NF-κB and AKT/mTOR signaling pathways and induces LC3B protein levels, resulting in autophagy. PA also increased the sub-G1 cell population that is characteristic of cell death. Therefore, the results of this study propose that PA inhibits HeLa cell viability through a mechanism mediated by the induction of autophagy. The study also suggests a new approach for cervical cancer therapeutics.

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.

Antitumor activity via apoptotic cell death pathway of water soluble copper(II) complexes: effect of the diamino unit on selectivity against lung cancer NCI-H460 cell line.

The cytotoxicity against five human tumor cell lines (THP-1, U937, Molt-4, Colo-205 and NCI-H460) of three water soluble copper(II) coordination compounds containing the ligands 3,3'-(ethane-1,2-diylbis(azanediyl))dipropanamide (BCEN), 3,3'-(piperazine-1,4-diyl)dipropanamide (BPAP) or 3,3'-and (1,4-diazepane-1,4-diyl)dipropanamide (BPAH) are reported in this work. The ligands contain different diamine units (ethylenediamine, piperazine or homopiperazine) and two propanamide units attached to the diamine centers, resulting in N2O2 donor sets. The complex containing homopiperazine unit presented the best antiproliferative effect and selectivity against lung cancer cell line NCI-H460, showing inhibitory concentration (IC50) of 58μmoldm-3 and Selectivity Index (SI) > 3.4. The mechanism of cell death promoted by the complex was investigated by Sub-G1 cell population analysis and annexin V and propidium iodide (PI) labeling techniques, suggesting that the complex promotes death by apoptosis. Transmission electron microscopy investigations are in agreement with the results presented by mitochondrial membrane potential analysis and also show the impairment of other organelles, including endoplasmic reticulum.