Apoptosis In Carcinoma Cells Research Articles

THEM6 is a prognostic biomarker for breast cancer and is associated with immune infiltration

To characterize the implications of lipid metabolism-related gene thioesterase superfamily member 6 (THEM6) in breast cancer. Several databases including The Cancer Genome Atlas (TCGA) were utilized for our meticulous bioinformatics analysis. We further performed qRT-PCR, immunoblotting and IHC assays to validate the expression of THEM6 in various breast cancer cells and tissues. In addition, we have carried out relevant functional experiments to explore the regulatory role of THEM6 in vitro. Lipid metabolism-related genes are independent factors for overall survival. According to several databases, THEM6 was significantly more expressed in cancerous tissues of breast invasive carcinoma (BRCA) compared to its paracancerous tissues. Furthermore, THEM6 overexpression was correlated with poorer overall survival of BRCA patients, serving as a separate prognostic factor for BRCA. Biological functional analyses revealed that THEM6 was associated with tumor progression and pathogenesis. Finally, we discovered that in BRCA, THEM6 expression was linked to multiple immune cell types. qRT-PCR and Western blotting experiments demonstrated a general upregulation of THEM6 expression in breast carcinoma cells. IHC showed that THEM6 was expressed in both breast cancer tissues and para-cancer tissues, but its expression level was significantly higher in carcinoma tissues. In vitro studies indicated that THEM6 increased proliferation, invasion, and inhibited apoptosis of breast carcinoma cells, while also affecting the cell cycle and promoting cancer progression. Furthermore, THEM6 may influence macrophage recruitment and polarization in the tumor microenvironment by regulating CCL2 secretion, which in turn affects macrophage recruitment in the tumor microenvironment. Our findings indicate that the overexpression of THEM6, which is linked to the development of breast cancer, is a predictor of a poor prognosis and has an impact on the degree of immune cell infiltration. Therefore, THEM6 has the potential to be a valuable target for BRCA.

Homoharringtonine induces apoptosis of mammary carcinoma cells by inhibiting the AKT/mTOR signaling pathway.

Mammary tumour is the most common type of tumour in dogs, especially in unneutered female dogs. Homoharringtonine (HHT) is a natural alkaloid that can be used to treat various types of human tumour. However, the inhibitory effect and mechanism of HHT on canine mammary carcinomas (CMC) remain unclear. This study aimed to evaluate the inhibitory effect of HHT on CMC in vitro and determine its underlying molecular mechanism. The effects of HHT on the cytotoxicity of CMC U27 cells were evaluated by the cell counting kit-8, wound healing, and Transwell assays. HHT-induced apoptosis of U27 cells was detected by JC-1 and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. Moreover, the gene expression of B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) were analysed using quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and the protein expression of protein kinase B/mammalian target of rapamycin (AKT/mTOR) and mitochondrial apoptosis proteins were determined by western blotting. Furthermore, mammary tumour-bearing mouse models were established using 4T1 cells to evaluate the therapeutic effect of HHT. It was found that HHT could significantly down-regulated the protein expression of p-AKT, p-mTOR, and Bcl-2, and up-regulated the protein expression of P53, Bax, cleaved caspase-3, and cleaved caspase-9. In addition, HHT significantly suppressed both tumour volume and mass in mammary tumour mice. In conclusion, HHT damages CMC cells by inhibiting the AKT/mTOR signalling pathway and inducing mitochondrial apoptosis. Such findings lay a theoretical foundation for the clinical treatment of CMC and provide more options for clinical medication.

A HIF-1α inhibitor combined with palmitic acid and L-carnitine treatment can prevent the fat metabolic reprogramming under hypoxia and induce apoptosis in hepatocellular carcinoma cells

BackgroundA hypoxic environment often persists within solid tumors, including hepatocellular carcinoma (HCC). Hypoxia-inducible factor-1α (HIF-1α) can accelerate cancer malignancy by inducing hypoxia-dependent expression of various genes. Tumor hypoxia can also induce metabolic reprogramming of fatty acid (FA) metabolism, through which HIF-1α plays an essential role in diminishing fatty acid β-oxidation (FAO) in hypoxic cancer cells.MethodsWe aimed to investigate potential new drug therapy options for targeting hypoxic cancer cells within HCC tumors, specifically through combining HIF-1α inhibition with palmitic acid (PA) + L-carnitine (LC) treatment to effectively induce apoptosis in hypoxic HCC cells. To test this hypothesis, in vitro and in vivo studies were performed.ResultsWe first demonstrated that hypoxia-dependent apoptosis was induced by an overload of PA in two HCC cell lines (HepG2 and Hep3B) via excessive production of reactive oxygen species (ROS). Moreover, this observed PA-induced apoptosis was enhanced by HIF-1α knockdown (KD) in these cells under hypoxia. In addition, the combination of PA with FAO activator LC increased FAO activity and led to stronger cell death than PA alone in hypoxic HIF-1α KD cells, specifically through further ROS generation. To clarify the mechanism of hypoxia-induced FA metabolism reprogramming, expression levels of the genes encoding FAO enzymes CPT1A, ACSL1, MCAD, and LCAD, FA transporter CD36, and FA esterification enzymes DGAT and APGAT were analyzed using HIF-1α KD and scramble control (SC) cells. The results suggested that HIF-1α could repress mRNA expression of the FAO-related enzymes and CD36, while it upregulated FA esterification gene expression. This suggested a central role for HIF-1α in hypoxia-induced reprogramming of FA metabolism in HCC cells. Using a nude mouse model, PA administration was found to induce apoptosis from ROS overproduction in HIF-1α KD tumors compared with SC tumors. Additional LC treatment synergistically enhanced the PA-induced apoptosis in HIF-1α KD tumors. Finally, in vivo therapy composed of HIF-1α inhibitor YC-1 with PA + LC could induce ROS-mediated apoptosis in HepG2 tumors without significant toxicity.ConclusionsA combination therapy of YC-1 with PA + LC may be a unique anti-tumor therapy for targeting hypoxic HCC cells, specifically by ROS overproduction leading to forced FAO activation.

Down-regulation of EZH2 genes targeting RUNX3 affects proliferation, invasion, and metastasis of human colon cancer cells by Wnt/β-catenin signaling pathway.

In order to detect the effect of EZH2 genes on proliferation, migration, invasion, and apoptosis of colon carcinoma cell strains HCT116 and HT29 by the Wnt/β-catenin signaling pathway, qRT-PCR was applied to measure relative expressions of EZH2, RUNX3, CEA, CA199, MMP-9, VEGF, β-catenin, and CyclinD1 in each group; Western-blot was employed with the intention of exploring relative expressions of these proteins in vivo and in vitro; monoclonal proliferation experiments and CCK-8 assay was adopted so as to check cell proliferation; the effect on cell migration was investigated via Transwell assay and cell scratch wound assay; flow cytometry was applied with a view to determining the effect on cell apoptosis. Transfected HCT116 cells are injected subcutaneously into nude mice. In colon cell strains HCT-116 and HT29, contrasted to the si-NC group, the RUNX3 expression was prominently up-regulated in the si-EZH2 group. Besides, expressions of CEA, CA199, MMP-9, and VEGF were significantly reduced; down-regulation of EZH2 genes remarkably inhibited cell proliferation, invasion and migration when facilitating apoptosis; down-regulation of EZH2 genes also significantly reduced expressions of essential proteins β-catenin and CyclinD1 on the Wnt pathway. The subcutaneous tumor body of nude mice was reduced. EZH2-OE is the opposite trend to si-EZH2; The EZH2 gene may target regulatory RUNX3 regulation via that Wnt/β-catenin signaling pathway, hence affecting colon carcinoma cell proliferation, invasion, migration, and apoptosis. Therefore, EZH2 may become a promising target for the clinical therapy of colon carcinoma.

POLE2 Regulates Apoptosis of Oral Squamous Cell Carcinoma Cells through the PI3K/AKT Signaling Pathway.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the head and neck, but its occurrence and progression mechanisms remain unclear. In addition-there is a lack of effective targeting drugs. The second major subunit of DNA polymerase (POLE2) catalyzes the prolongation of new strand replication and modifies exonuclease domain activity. Our previous study found that POLE2 was associated with OSCC progression, but the mechanism remains unclear. The expression of POLE2 in OSCC tissues was detected using immunological assays. Mann-Whitney U analysis was used to investigate the relationship between POLE2 gene expression and tumor classification and prognosis of OSCC. POLE2 expression was inhibited in OSCC cells, and the effects of gene and protein expression were detected using RT-PCR and Western blotting. The POLE2 knockout model was constructed by transfecting a lentiviral vector. Cell proliferation, apoptosis, and migration were detected using various assays including colony formation, MTT, flow cytometry, wound healing assay, Transwell assay, and the Human Apoptosis Antibody Array. The animal model of OSCC was established by subcutaneous injection of transfected HN6 into 4-week-old female nude mice. After 30 days, tumors were removed under anesthesia and tumor weight and dimension were recorded. Tumor cell proliferation was analyzed using Ki67 staining. POLE2 gene levels were significantly higher in the OSCC tissues than in the normal tissues. In addition, POLE2 gene levels were statistically correlated with tumor classification and prognosis. Silencing POLE2 inhibited the proliferation of oral cancer cells and promoted apoptosis in vitro. Animal experiments also supported a positive correlation between POLE2 and OSCC tumor formation. We further demonstrated that POLE2 could upregulate the expression of apoptosis-related proteins such as caspase-3, CD40, CD40L, DR6, Fas, IGFBP-6, p21, and SMAC. In addition, POLE2 regulated OSCC development by inhibiting the PI3K/AKT signaling pathway. POLE2 is closely related to the progression of OSCC. Thus, POLE2 may be a potential target for OSCC treatment.

Homospisulosine induced apoptosis in cervical carcinoma cells is associated with phosphorylation of Bcl-2 and up-regulation of p27/Kip1.

Spisulosine (1-deoxysphinganine) is a sphingoid amino alcohol isolated from the sea clams that showed potent antiproliferative activity against a broad spectrum of solid tumors but failed in clinical trials due to neurotoxicity. However, its structural similarity to other bioactive sphingoids, interesting mode of action, and appreciable potency against cancer cells make it a suitable lead for future anticancer drug development. The present study was conducted to elucidate mechanisms of the antiproliferative/cytotoxic effects of newly synthesized spisulosine analog homospisulosine (KP7). The evaluation was performed on cervical carcinoma cells, representing an in vitro model of one of the most common cancer types and a significant worldwide cause of women's cancer mortality. Treatment with homospisulosine (2.0 μM) for 24, 48, and 72 h significantly inhibited the growth of HeLa cells in vitro and induced apoptosis detectable by DNA fragmentation, externalization of phosphatidylserine, dissipation of mitochondrial membrane potential, activation of caspase-3 and cleavage of PARP. In addition, treating HeLa cells with spisulosine increased p27 and Bcl-2 on protein levels and phosphorylation of Bcl-2 on Ser70 residue. These results support the potential for spisulosine analogs represented here by homospisulosine for future therapeutic development.

2α, 3α, 24-Thrihydroxyurs-12-en-24-ursolic acid enhances the cytotoxic effect of cisplatin on oral cancer cells by down-regulating autophagy.

This study aimed to investigate the effect and mechanism of 2α, 3α, 24-thrihydroxyurs-12-en-24-ursolic acid (TEOA) alone or in combination with cisplatin on oral cancer. TEOA, a pentacyclic triterpenoid compound isolated from the roots of Actinidia eriantha, has demonstrated antitumor activity in preclinical experiments. However, its role in oral cancer remains poorly understood. Our findings revealed that a low concentration of TEOA did not exhibit significant cytotoxicity against oral squamous cell carcinoma cells. However, when combined with cisplatin, TEOA showed a significant therapeutic effect. The combined treatments resulted in a significant inhibition of proliferation and migration and a significant increase in apoptosis of squamous cell carcinoma cells. Cisplatin exposure increased autophagy levels, which may contribute to chemoresistance. Of note, the presence of TEOA significantly inhibited cisplatin-induced autophagy, leading to improved chemotherapy efficacy. Our findings indicate that a mild low dosage of TEOA may enhance the cytotoxic effect of cisplatin by downregulating autophagy in oral cancer cells.

Potential ‘anti-cancer’ effects of esketamine on proliferation, apoptosis, migration and invasion in esophageal squamous carcinoma cells

BackgroundEsketamine, an N-methyl-D-aspartate receptor antagonist, is commonly used for anesthesia and analgesia clinically. It was reported to negatively regulate cell proliferation, metastasis and apoptosis in cancer cells, including lung cancer and pancreatic cancer. However, its impact on esophageal squamous cell carcinoma (ESCC) malignance and underlying mechanism remain elusive. This study was aimed to investigate the antitumor effects of esketamine on ESCC in vitro.MethodsESCC cell lines (KYSE-30 and KYSE-150) were cultured and treated with different concentrations (0.1, 0.2, 0.4, 0.8, 1, 2 mM) of esketamine. Their proliferation, apoptosis, migration and invasion were assessed with various assays. Furthermore, mass spectrometry-based proteomic analysis and GO/KEGG enrichment analysis were applied to characterize the differentially expressed proteins (DEPs) with or without esketamine treatment. Some key proteins identified from proteomic analysis were further validated with Western blotting and bioinformatics analysis.ResultsEsketamine significantly inhibited the proliferation, migration, invasion and promoted apoptosis of the both types of cell lines in a dose- and time-dependent manner. A total of 321 common DEPs, including 97 upregulated and 224 downregulated proteins, were found with HPLC–MS analyses. GO/KEGG enrichment analysis suggested that esketamine affected cell population proliferation, GTPase activity and Apelin signaling pathway. The ERCC6L, AHR and KIF2C protein expression was significantly downregulated in these ESCC cells treated with esketamine compared to the controls and their changes were associated with the suppressive effects of esketamine on ESCC through bioinformatics analysis.ConclusionsOur work demonstrated that esketamine has potential anti-ESCC properties in vitro but subjected to further in vivo and clinical study.

Evaluation of the antitumoral effects of the mesoionic compound MI-D: Implications for endothelial cells viability and angiogenesis inhibition

Angiogenesis is considered one of the hallmarks of cancer, assisting tumor progression and metastasis. The mesoionic compound, MI-D, can induce cell death and provoke cytoskeletal and metabolic changes in cancer cells. Using in vitro and in vivo models, this study aimed to evaluate the effects of MI-D on the viability of human endothelial cells (EC) and its ability to inhibit tumor-induced angiogenesis induced by tumoral cells. For in vitro analysis, colon carcinoma (HT29) and endothelial (EA.hy926) cells were used as the tumoral and angiogenesis models, respectively. To evaluate cytotoxicity, methylene blue viability stain and annexin-V/7AAD tests were performed with both cell types. For the angiogenesis experiments, scratch wound healing and capillary tube-like formation assays were performed with the EC. The in vivo tests were performed with the chorioallantoic membrane (HET-CAM) methodology, wherein gelatin sponge implants containing MI-D (5, 25, and 50 μM), HT29 cells, or both were grafted in the CAM. Our data showed that MI-D induced apoptosis in both endothelial and colon carcinoma cells, with a strong cytotoxic effect on the tumoral lineage. The drug inhibited the EC's migration and capillary-like structure formation in vitro. In the HET-CAM assays, MI-D reduced the number of blood vessels in the membrane when grafted alone and accompanied by tumor cells. In this study, MI-D interfered in important steps of angiogenesis, such as maintenance of endothelial cell viability, migration, formation of capillary-like structures, as well tumor-induced neovascularization, reinforcing the hypothesis that MI-D might act as an inhibitor of angiogenesis, and a potential antitumor agent.

The Secondary Metabolites of Bacillus subtilis Strain Z15 Induce Apoptosis in Hepatocellular Carcinoma Cells.

The lipopeptides produced by Bacillus subtilis have anti-cancer potential. We had previously identified a secondary metabolite of B. subtilis strain Z15 (BS-Z15), which has an operon that regulates lipopeptide synthesis, and also demonstrated that the fermentation products of this strain exerted antioxidant and pro-immune effects. The purpose of this study was to investigate in vitro and in vivo the anticancer effects of BS-Z15 secondary metabolites (BS-Z15 SMs) on hepatocellular carcinoma (HCC) cells. BS-Z15 SMs significantly inhibited H22 cell-derived murine xenograft tumor growth without any systemic toxicity. In addition, BS-Z15 SMs decreased the viability of H22 cells and BEL-7404 cells in vitro with respective IC50 values of 33.83 and 27.26µg/mL. Consistent with this, BS-Z15 SMs induced apoptosis and G0/G1 phase arrest in the BEL-7404 cells, and the mitochondrial membrane potential was also significantly reduced in a dose-dependent manner. Mechanistically, BS-Z15 SMs upregulated the pro-apoptotic p53, Bax, cytochrome C, and cleaved-caspase-3/9 proteins and downregulated the anti-apoptotic Bcl-2. These findings suggest that the induction of apoptosis in HCC cells by BS-Z15 SMs may be related to the mitochondrial pathway. Thus, the secondary metabolites of B. subtilis strain Z15 are promising to become new anti-cancer drugs for the clinical treatment of liver cancer.

Attenuating Colorectal Cancer Using Nine Cultivars of Australian Lupin Seeds: Apoptosis Induction Triggered by Mitochondrial Reactive Oxygen Species Generation and Caspases-3/7 Activation.

As Australian lupin cultivars are rich sources of polyphenols, dietary fibers, high-quality proteins, and abundant bioactive compounds with significant antioxidant, antidiabetic, and anticancer activities, this research work is aimed at investigating the colon cancer alleviation activity of nine cultivars of lupin seeds on HCT116 and HT29 colon carcinoma cell lines through anti-proliferation assay, measurement of apoptosis, and identification of the mechanism of apoptosis. Nine cultivars were pre-screened for anti-proliferation of HCT116 and HT29 cells along with consideration of the impact of heat processing on cancer cell viability. Mandelup and Jurien showed significant inhibition of HCT116 cells, whereas the highest inhibition of HT29 cell proliferation was attained by Jurien and Mandelup. Processing decreased the anti-proliferation activity drastically. Lupin cultivars Mandelup, Barlock, and Jurien (dose: 300 μg/mL) induced early and late apoptosis of colon cancer cells in Annexin V-FITC assay. The mechanism of apoptosis was explored, which involves boosting of caspases-3/7 activation and intracellular reactive oxygen species (ROS) generation in HCT116 cells (Mandelup and Barlock) and HT29 cells (Jurien and Mandelup). Thus, the findings showed that lupin cultivars arrest cell cycles by inducing apoptosis of colorectal carcinoma cells triggered by elevated ROS generation and caspases-3/7 activation.

Dcalycinumines A—E, Alkaloids with Cytotoxic Activities of Nasopharyngeal Carcinoma Cells from Daphniphyllum calycinum

Comprehensive SummaryFive novel Daphniphyllum alkaloids, named dcalycinumines A—E (1—4, 6), and eight previously described Daphniphyllum alkaloids (5, 7—13) were isolated from Daphniphyllum calycinum. Compound 1 is the first Daphniphyllum alkaloid possessing a highly rearranged 6/6/6/7/5/6 hexacyclic architecture with a unique 3‐methyl‐1‐azabicyclo [4,4,0] decane ring system. Compound 2 represents a rare diamino Daphniphyllum alkaloid with an unprecedented 6/5/5/6/6/5 carbon skeleton featuring a unique 1‐aza‐6‐azaspiro [4,5] decane unit, whereas 3 also represents a rare diamino Daphniphyllum alkaloid as a possible precursor of 2. Compound 4 is the second example of C‐22‐nor yuzurimine‐type alkaloids. Their structures and absolute configurations were elucidated by HRESIMS, NMR spectroscopic analyses, ECD calculations, and single‐crystal X‐ray diffraction. Moreover, compound 1 showed remarkable antitumor activities, which could inhibit the proliferation, migration and invasion of nasopharyngeal carcinoma cells, and promoted nasopharyngeal carcinoma cells apoptosis.

GABA regulates the proliferation and apoptosis of head and neck squamous cell carcinoma cells by promoting the expression of CCND2 and BCL2L1

Gamma-aminobutyric acid (GABA) is a multifunctional molecule that is widely present in the nervous system and nonneuronal tissues. It plays pivotal roles in neurotransmission, regulation of secretion, cell differentiation, proliferation, and tumorigenesis. However, the exact mechanisms of GABA in head and neck squamous cell carcinomas (HNSCCs) are unknown. We took advantage of RNA sequencing in this work and uncovered the potential gene expression profiles of the GABA-treated HNSCC cell line HN4–2. We found that the expression of CCND2 and BCL2L1 was significantly upregulated. Furthermore, GABA treatment inhibited the cell apoptosis induced by cisplatin and regulated the cell cycle after treatment with cisplatin in HN4–2 cells. Moreover, we also found that GABA could upregulate the expression of CCND2 and BCL2L1 after treatment with cisplatin. Our results not only reveal the potential pro-tumorigenic effect of GABA on HNSCCs but also provide a novel therapeutic target for HNSCC treatment.

Escin-sorafenib synergy up-regulates LC3-II and p62 to induce apoptosis in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a common solid cancer and the leading cause of cancer deaths worldwide. Sorafenib is the first drug used to treat HCC but its effectiveness needs to be improved, and it is important to find ways to treat cancer that combine sorafenib with other drugs. Synergistic therapies lower effective drug doses and side effects while enhancing the anticancer effect. In the present study, the therapeutic potential of sorafenibin combination with escin and its underlying mechanism in targeting liver cancer has been established. The IC50 of sorafenib and escin against HepG2, PLC/PRF5 and Huh7 cell lines were determined using MTT assay. The combination index, dose reduction index, isobologram and concentrations producing synergy were evaluated using the Chou-Talaly algorithm. The sub-effective concentration of sorafenib and escin was selected to analyze cytotoxic synergistic potential. Cellular ROS, mitochondrial membrane potential, annexin V and cell cycle were evaluated using a flow-cytometer, and autophagy biomarkers were determined using western blotting. Moreover, autophagy was knocked down using ATG5 siRNA to confirm its role. A DEN-induced liver cancer rat model was developed to check the synergy of sorafenib and escin. Different concentrations of escin reduced the IC50 of sorafenib in HepG2, PLC/PRF5 and Huh7 cell lines. Chou-Talaly algorithm determined cytotoxic synergistic concentrations of sorafenib and escin in these cell lines. Mechanistically, this combination over-expressed p62 and LC-II, reflecting autophagy block and induced late apoptosis, further reconfirmed by ATG5 knockdown. Sorafenib and escin combination reduced HCC serum biomarker α-feto protein (α-FP) by 1.5 folds. This combination restricted liver weight, tumor number and size, also, conserved morphological features of liver cells. The combination selectively targeted the G0 /G1 phase of cancer cells. Escin and sorafenib combination potentially up-regulates p62 to block autophagy to induce late apoptosis in liver cancer cells.

Artemetin, A Dietary Flavonoid Inhibits the Proliferation and Induces Apoptosis in Human Gastric Carcinoma (AGS) Cells

Artemetin, A Dietary Flavonoid Inhibits the Proliferation and Induces Apoptosis in Human Gastric Carcinoma (AGS) Cells

Effects of ceramide C2 application on human laryngeal carcinoma cells: a cell culture study.

In the present study, we investigated the effects of Ceramide C2 application on human laryngeal carcinoma cells. Human larynx epidermoid carcinoma HEp-2 (ATCC® CCL-23™) cells were purchased from the American Type Culture Collection (ATCC, USA). Human larynx epidermoid carcinoma HEp-2 cells were cultured in complete Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS) (10%) and penicillin/streptomycin (1%) in a CO2 (5%) incubator under standard cell culture conditions. Ceramide C2 was prepared, and further dilutions ranging from 3.13 to 100 μM were prepared in a fresh culture medium. Cells on 96 well plates were exposed to the prepared concentrations of ceramide C2 for 24 and 48 hours. Cytotoxicity evaluation was performed by MTT. Apoptosis profiles of HEp-2 cells were detected by annexin-V analysis. The activated caspases 3/7 on HEp-2 cells after ceramide C2 exposure were evaluated with flow cytometric analysis. The morphological changes on HEp-2 cells caused by ceramide C2 were evaluated by staining with phalloidine and acridine orange via confocal microscopy. For the Wound Healing Assay, HEp-2 cells were cultured in 6 well-plates until they became confluent. MTT cytotoxicity test findings revealed that the viability of human laryngeal carcinoma cells decreased with the increased application of ceramide C2 for 24 hours compared to untreated (control) cells. The highest growth inhibition by ceramide C2 for short-term application for 24 hours was detected at the highest concentration of ceramide C2 (100 µM). Annexin-V findings showed that 98.97 of HEp-2 cells were alive, and 1.63% were detected as early apoptosis for the control group. The results showed that ceramide C2 triggered apoptosis on HEp-2 cells with a percentage of total apoptotic cells of 61,40 compared to untreated HEp-2 cells. Cysteine proteases (caspases) 3/7 activation percentages of HEp-2 cells exposed to ceramide C2 for 24 hours were compared to control cells, and the morphology of HEp-2 cells was changed with clear apoptotic signs that underlined the cytotoxicity and pro-apoptotic activity of ceramide C2. Scratch Assay assessed the migration capability of HEp-2 cells before and after the exposure to ceramide C2. It showed that ceramide C2 reduced human laryngeal carcinoma cells' migration capability and proliferation for 24 hours. Based on all study findings, it can be considered that short-chain ceramide C2 exerted cytotoxicity on human laryngeal carcinoma cells in a dose and time-dependent manner and reduced the viability via inducing caspase-dependent apoptosis. The overall effect might be derived from the elevated intracellular ceramide levels by the exogenous application of ceramide C2. Consequently, it was concluded that ceramide C2 has good potential to cause cytotoxicity and apoptosis in human laryngeal carcinoma cells and, after deeper in vitro and in vivo investigations, can be a good candidate for designing anti-cancer drugs with high efficiency.

Solamargine induces apoptosis of human renal carcinoma cells via downregulating phosphorylated STAT3 expression.

Solamargine (SM), an active compound derived from Solanum nigrum, triggers apoptosis and inhibits the metastatic and oxidative activities of various types of tumor cells. However, the effect of SM on human renal carcinoma cells remains unknown. In the present study, the molecular mechanisms underlying the antitumor effects of SM on ACHN and 786-O cells were elucidated. Specifically, MTT and colony formation assays were conducted to evaluate the impact of SM treatment on the proliferation of ACHN and 786-O cells, and flow cytometry was conducted to determine the influence of SM on the apoptosis rates of these cells. In addition, the expression of target proteins was determined by western blotting. The results revealed that SM not only inhibited cell viability but also promoted the apoptosis of ACHN and 786-O cells in a time- and dose-dependent manner. Moreover, treatment of ACHN and 786-O cells with SM significantly enhanced the caspase-3, caspase-8 and caspase-9 activities. Furthermore, SM downregulated the expression of phosphorylated signal transducer and activator of transcription-3 (p-STAT3) and Bcl-2 but increased the expression of cleaved caspase-3, -8, -9 and Bax. BAY2353, a p-STAT3 inhibitor, inhibited the viability of ACHN and 786-O cells, increased the expression of cleaved caspase-9 and Bax and decreased the expression of p-STAT3 and Bcl-2. Further experiments demonstrated that SM inhibited tumor growth in xenograft nude mice without causing specific toxicity to the major organs. Collectively, these findings indicated that SM not only inhibited the viability but also promoted the apoptosis of ACHN and 786-O cells, through a mechanism involving downregulation of p-STAT3 expression.

Milk thistle nano-micelle formulation promotes cell cycle arrest and apoptosis in hepatocellular carcinoma cells through modulating miR-155-3p /SOCS2 /PHLDA1 signaling axis

BackgroundHepatocellular Carcinoma (HCC) is a prevalent form of liver cancer that causes significant mortality in numerous individuals worldwide. This study compared the effects of milk thistle (MT) and nano-milk thistle (N-MT) on the expression of the genes that participate in apoptosis and cell cycle pathways in Huh-7 and HepG2 cells.MethodsIC50 values of MT and N-MT were determined using the MTT assay. Huh-7 and HepG2 cell lines (containing mutant and wild-type TP53 gene, respectively) were incubated with MT and N-MT for 24h and 48h and the impact of MT and N-MT on the proliferation of these cell lines was evaluated through a comparative analysis. Cell cycle and apoptosis were assessed by flow cytometry after 24h and 48h treatment in the cell lines mentioned. Real-time PCR was used to analyze miR-155-3p, PHLDA1, SOCS2, TP53, P21, BAX, and BCL-2 expression in the cell lines that were being treated.ResultsN-MT reduces cancer cell growth in a time and concentration-dependent manner, which is more toxic compared to MT. Huh-7 was observed to have IC50 values of 2.35 and 1.7 μg/ml at 24h and 48h, and HepG2 was observed to have IC50 values of 3.4 and 2.6 μg/ml at 24 and 48h, respectively. N-MT arrested Huh-7 and HepG2 cells in the Sub-G1 phase and induced apoptosis. N-MT led to a marked reduction in the expression of miR-155-3p and BCL-2 after 24h and 48h treatments. Conversely, PHLDA1, SOCS2, BAX, and P21 were upregulated in the treated cells compared to untreated cells, which suggests that milk thistle has the potential to regulate these genes. N-MT reduced the expression of TP53 in Huh-7 cells after mentioned time points, while there was a significant increase in the expression of the TP53 gene in HepG2 cells. No gene expression changes were observed in MT-treated cells after 24h and 48h.ConclusionN-MT can regulate cancer cell death by arresting cell cycle and inducing apoptosis. This occurs through the alteration of apoptotic genes expression. A reduction in the expression of miR-155-3p and increase in the expression of SOCS2 and PHLDA1 after N-MT treatment showed the correlation between miR-155-3p and PHLDA1/SOCS2 found in bioinformatics analysis. While N-MT increased TP53 expression in HepG2, reduced it in Huh-7. The findings indicate that N-MT can function intelligently in cancer cells and can be a helpful complement to cancer treatment.

Low glucose metabolite 3-phosphoglycerate switches PHGDH from serine synthesis to p53 activation to control cell fate.

Glycolytic intermediary metabolites such as fructose-1,6-bisphosphate can serve as signals, controlling metabolic states beyond energy metabolism. However, whether glycolytic metabolites also play a role in controlling cell fate remains unexplored. Here, we find that low levels of glycolytic metabolite 3-phosphoglycerate (3-PGA) can switch phosphoglycerate dehydrogenase (PHGDH) from cataplerosis serine synthesis to pro-apoptotic activation of p53. PHGDH is a p53-binding protein, and when unoccupied by 3-PGA interacts with the scaffold protein AXIN in complex with the kinase HIPK2, both of which are also p53-binding proteins. This leads to the formation of a multivalent p53-binding complex that allows HIPK2 to specifically phosphorylate p53-Ser46 and thereby promote apoptosis. Furthermore, we show that PHGDH mutants (R135W and V261M) that are constitutively bound to 3-PGA abolish p53 activation even under low glucose conditions, while the mutants (T57A and T78A) unable to bind 3-PGA cause constitutive p53 activation and apoptosis in hepatocellular carcinoma (HCC)cells, even in the presence of high glucose. In vivo, PHGDH-T57A induces apoptosis and inhibits the growth of diethylnitrosamine-induced mouse HCC, whereas PHGDH-R135W prevents apoptosis and promotes HCC growth, and knockout of Trp53 abolishes these effects above. Importantly, caloric restriction that lowers whole-body glucose levels can impede HCC growth dependent on PHGDH. Together, these results unveil a mechanism by which glucose availability autonomously controls p53 activity, providing a new paradigm of cell fate control by metabolic substrate availability.

Let-7g Upregulation Attenuated the KRAS-PI3K-Rac1-Akt Axis-Mediated Bioenergetic Functions.

The aberrant activation of signaling pathways contributes to cancer cells with metabolic reprogramming. Thus, targeting signaling modulators is considered a potential therapeutic strategy for cancer. Subcellular fractionation, coimmunoprecipitation, biochemical analysis, and gene manipulation experiments revealed that decreasing the interaction of kirsten rat sarcoma viral oncogene homolog (KRAS) with p110α in lipid rafts with the use of naringenin (NGN), a citrus flavonoid, causes lipid raft-associated phosphatidylinositol 3-kinase (PI3K)-GTP-ras-related C3 botulinum toxin substrate 1 (Rac1)-protein kinase B (Akt)-regulated metabolic dysfunction of glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), leading to apoptosis in human nasopharyngeal carcinoma (NPC) cells. The use of lethal-7g (let-7g) mimic and let-7g inhibitor confirmed that elevated let-7g resulted in a decrease in KRAS expression, which attenuated the PI3K-Rac1-Akt-BCL-2/BCL-xL-modulated mitochondrial energy metabolic functions. Increased let-7g depends on the suppression of the RNA-specificity of monocyte chemoattractant protein-induced protein-1 (MCPIP1) ribonuclease since NGN specifically blocks the degradation of pre-let-7g by NPC cell-derived immunoprecipitated MCPIP1. Converging lines of evidence indicate that the inhibition of MCPIP1 by NGN leads to let-7g upregulation, suppressing oncogenic KRAS-modulated PI3K-Rac1-Akt signaling and thereby impeding the metabolic activities of aerobic glycolysis and mitochondrial OXPHOS.