Age-associated features of the expression level of apoptosis markers in cardiomyocytes of patients with dilated cardiomyopathy

Для понимания патогенеза дилатационной кардиомиопатии (ДКМП) необходимо установить молекулярно-клеточные механизмы старения миокарда, в том числе связанные с программируемой клеточной гибелью, молекулярные механизмы которого практически не изучены. Цель работы - изучение маркеров апоптоза в кардиомиоцитах у пациентов с ДКМП in vitro. В работе использовали метод первичных диссоциированных клеточных культур и метод иммунофлюоресцентной конфокальной лазерной микроскопии. Для моделирования клеточного старения использовали клетки 3-го и 14-го пассажей, соответствующие «молодым» и «старым» культурам. На молекулярном уровне старение клеток кардиомиоцитов сопровождалось повышением экспрессии р16 в 2 раза по сравнению с «молодыми культурами» как в контрольной, так и в группе с ДКМП. Также установлено, что экспрессия р16 в культурах, взятых от пациентов с патологией, была в 2 раза выше, чем в аналогичных культурах от здоровых пациентов. Экспрессия р21 была повышена в группе с ДКМП по сравнению с контрольной группой, однако при старении культуры экспрессия p21 не изменялась, оставаясь на высоком уровне. Наиболее значимые различия были получены при сравнении экспрессии Bax в культуре клеток кардиомиоцитов из группы с ДКМП в «молодой» культуре с нормой - в 3,2 раза. Старение клеток миокарда на молекулярном уровне проявлялось в повышении экспрессии белка Baх, именно он является запускающим механизмом митохондриального пути апоптоза. Возможно, этот путь клеточной гибели является превалирующем при ДКМП. To understand the pathogenesis of dilated cardiomyopathy (DCMP), it is necessary to establish the molecular-cellular mechanisms of myocardial aging, including those associated with programmed cell death, the molecular mechanisms of which have not been practically studied. The aim of this work is to study markers of apoptosis in cardiomyocytes of patients with DCMP in vitro. We used the method of primary dissociated cell cultures and the method of immunofluorescence confocal laser microscopy. Cells of the 3 and 14 passages, corresponding to «young» and «old» cultures, were used to simulate cellular senescence. Results. At the molecular level, aging of cardiomyocyte cells was accompanied by a twofold increase in the expression of p16 compared to «young cultures» both in the control group and in the group with DCMP. It was also found that the expression of p16 in cultures taken from patients with pathology was 2 times higher than in similar cultures from healthy patients. The expression of p21 was increased in the group with DCMP compared to the control; however, with aging of the culture, the expression of p21 did not change, remaining at a significant level. The most significant differences were obtained when comparing the expression of Bax in the cell culture of cardiomyocytes from the group with DCMP in a «young» culture compared with the norm, 3,2 times. Aging of myocardial cells at the molecular level was manifested in an increase in the expression of the Bax protein, which is the triggering mechanism of the mitochondrial apoptosis pathway. It is possible that this pathway of cell death is prevalent in DCMP.

MED10 Drives the Oncogenicity and Refractory Phenotype of Bladder Urothelial Carcinoma Through the Upregulation of hsa-miR-590

BackgroundDysfunctional transcription machinery with associated dysregulated transcription characterizes many malignancies. Components of the mediator complex, a principal modulator of transcription, are increasingly implicated in cancer. The mediator complex subunit 10 (MED10), a vital kinase module of the mediator, plays a critical role in bladder physiology and pathology. However, its role in the oncogenicity, metastasis, and disease recurrence in bladder cancer (BLCA) remains unclear.ObjectiveThus, we investigated the role of dysregulated or aberrantly expressed MED10 in the enhanced onco-aggression, disease progression, and recurrence of bladder urothelial carcinoma (UC), as well as the underlying molecular mechanism.MethodsUsing an array of multi-omics big data analyses of clinicopathological data, in vitro expression profiling and functional assays, and immunocytochemical staining, we assessed the probable roles of MED10 in the progression and prognosis of BLCA/UC.ResultsOur bioinformatics-aided gene expression profiling showed that MED10 is aberrantly expressed in patients with BLCA, is associated with high-grade disease, is positively correlated with tumor stage, and confers significant survival disadvantage. Reanalyzing the TCGA BLCA cohort (n = 454), we showed that aberrantly expressed MED10 expression is associated with metastatic and recurrent disease, disease progression, immune suppression, and therapy failure. Interestingly, we demonstrated that MED10 interacts with and is co-expressed with the microRNA, hsa-miR-590, and that CRISPR-mediated knockout of MED10 elicits the downregulation of miR-590 preferentially in metastatic UC cells, compared to their primary tumor peers. More so, silencing MED10 in SW1738 and JMSU1 UC cell lines significantly attenuates their cell proliferation, migration, invasion, clonogenicity, and tumorsphere formation (primary and secondary), with the associated downregulation of BCL-xL, MKI67, VIM, SNAI1, OCT4, and LIN28A but upregulated BAX protein expression. In addition, we showed that high MED10 expression is a non-inferior biomarker of urothelial recurrence compared with markers of cancer stemness; however, MED10 is a better biomarker of local recurrence than any of the stemness markers.ConclusionThese data provide preclinical evidence that dysregulated MED10/MIR590 signaling drives onco-aggression, disease progression, and recurrence of bladder UC and that this oncogenic signal is therapeutically actionable for repressing the metastatic/recurrent phenotypes, enhancing therapy response, and shutting down stemness-driven disease progression and relapse in patients with BLCA/UC.

Neuroprotective effects of Lippia javanica (Burm.F.) Spreng. Herbal tea infusion on Lead-induced oxidative brain damage in Wistar rats

Background Though Lippia javanica (Burm.f.) Spreng antioxidant activity has been demonstrated, its effect in protecting the brain from lead (Pb)-induced oxidative damage is unknown. This study investigated the effect of L. javanica against Pb-induced oxidative stress, inflammation, apoptosis and acetylcholinesterase activity in rat’s brain. Methods L. javanica herbal tea infusion was prepared, its phytochemical constituent was revealed by liquid chromatography-Mass spectrometer (LC-MS) and was administered simultaneously with Pb. Four groups of male Wistar rats (n = 5/group) were used: control received distilled water; Pb-acetate group received 50 mg Pb/ Kg bodyweight (bw), treatment group received 50 mg Pb/ Kg Pb-acetate + 5 ml/kg bw L. javanica and L. javanica group received 5 ml/Kg bw of L. javanica tea infusion only. After 6 weeks of treatment, oxidative status, acetylcholinesterase activity, inflammation and apoptosis was assessed in brain tissue which was also histologically examined. Results Mean brain and heart weight was reduced (p < 0.05) while liver and spleen weights were increased (p < 0.05) in Pb exposed animals but were prevented by L. juvanica treatment. Treatment with L. javanica increased (p < 0.05) overall brain antioxidant status (glutathione and superoxide dismutase activities) and reduced lipid peroxidation (p < 0.05) compared to the Pb exposed animals. Pro-inflammatory cytokine tumour necrotic factor-alpha, pro-apoptosis Bax protein and anticholinesterase activity were reduced (p < 0.05) in Pb-L. javanica treated animals compared to the Pb exposed group. Histological examination confirmed neuroprotective effects of L. javanica as evidenced by reduced apoptosis/necrosis and inflammation-induced vacuolization and oedema in the hippocampus. The L. javanica treatment alone had no detrimental effects to the rats. LC-MS analysis revealed L. javanica to be rich in phenolics. Conclusions This study demonstrated that L. javanica, rich in phenolics was effective in reducing Pb-induced brain oxidative stress, inflammation, apoptosis, acetylcholinesterase activity and neuronal damage.

Association Study of BIF-1 Gene Expression with Histopathological Characteristics and Hormone Receptors in Breast Cancer

Background Breast cancer (BC) is a heterogeneous disease that has different clinical outcomes. Bax-interacting Factor-1 (BIF-1) is a member of the endophilin B family that produces the pro-apoptotic BCL2-Associated X (BAX) protein in response to apoptotic signals. Lack of BIF-1 inhibits the intrinsic pathway of apoptosis and increases the risk of tumor genesis. The aim of the present study was to investigate the relationship between hormone receptors (ER, PR, HER2) status and different levels of BIF-1 gene expression in breast cancer patients. Methods BIF-1 gene expression was evaluated in 50 breast cancer tumors and 50 normal breast mammary tissues using SYBR Green Real Time RT-PCR technique. Multivariate and univariate analyses were used to evaluate the relationship between the prognostic significance of the BIF-1 gene using SPSS software. In this study, BIF-1 was selected as a candidate for a molecular biomarker and its expression status in breast cancer patients with hormone receptors (ER, RR, HER2) compared to patients without these hormone receptors. Results The study showed that the relative expression of BIF-1 gene in tissues of patients with hormone receptor in breast cancer compared to those without hormone receptor were not statistically significant. The expression levels of BIF-1 gene in different groups were evaluated for hormone receptor status. No significant relationship was found between BIF-1 gene expression and hormone receptors (ER, PR and HER2) (p> 0.05). Conclusion BIF-1 gene expression may be a useful prognostic marker in breast cancer.

Growth Inhibitory Efficacy of Chinese Herbs in a Cellular Model for Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is characterized by the absence of estrogen receptor-α progesterone receptor and human epidermal growth factor receptor-2. Treatment for this breast cancer subtype is restricted to multidrug chemotherapy and survival pathway-based molecularly targeted therapy. The long-term treatment options are associated with systemic toxicity, spontaneous and/or acquired tumor resistance and the emergence a of drug-resistant stem cell population. These limitations lead to advanced stage metastatic cancer. Current emphasis is on research directions that identify efficacious, naturally occurring agents representing an unmet need for testable therapeutic alternatives for therapy resistant breast cancer. Chinese herbs are widely used in traditional Chinese medicine in women for estrogen related health issues and also for integrative support for cancer treatment. This review discusses published evidence on a TNBC model for growth inhibitory effects of several mechanistically distinct nontoxic Chinese herbs, most of them nutritional in nature, and identifies susceptible pathways and potential molecular targets for their efficacy. Documented anti-proliferative and pro-apoptotic effects of these herbs are associated with downregulation of RB, RAS, PI3K, and AKT signaling, modulation of Bcl-2/BAX protein expressions and increased caspase activity. This review provides a proof of concept for Chinese herbs as testable alternatives for prevention/therapy of TNBC.

Response of MCF-7 Breast Cancer Cells Overexpressed with P-Glycoprotein to Apoptotic Induction after Photodynamic Therapy

Multidrug resistance (MDR) has posed a significant threat to cancer treatment and has led to the emergence of a new therapeutic regime of photodynamic therapy (PDT) to curb the menace. The PDT modality employs a photosensitiser (PS), excited at a specific wavelength of light to kill cancer cells. In the present study, we used a zinc phthalocyanine tetrasulfonic acid PS to mediate the photodynamic killing of MCF-7 cells overexpressed with P-glycoprotein (P-gp) and investigate the response to cell death induction. After photodynamic treatment, MCF-7 cells undergo cell death, and indicators like Annexin V/PI staining, DNA fragmentation, and measurement of apoptotic protein expression were investigated. Results showed increased externalisation of phosphatidylserine protein, measured as a percentage in flow cytometry indicative of apoptotic induction. This expression was significant (p < 0.006) for the untreated control cells, and there was no detection of DNA fragments after a laser fluence of 20 J/cm2. In addition, a statistically significant difference (p < 0.05) was seen in caspase 8 activity and Bax protein expression. These findings were indicative of apoptotic induction and thus seem to represent the extrinsic apoptotic pathway. This study shows the role of PDT in the treatment of a resistant phenotype breast cancer.

Antiproliferative and cytotoxic activity of Geraniaceae plant extracts against five tumor cell lines

Aim: To determine the antiproliferative and cytotoxic activities of Geranium and Erodium species against human cancer and noncancer cell lines, respectively. Methods: Twenty-one species of Geranium and Erodium were extracted and screened against cancerous and noncancerous human cell lines. Results: In a dose-response manner, G. glaberrimum, G. asphodeloides, E. brandianum and E. leucanthum were able, with variable potency, to inhibit cellular proliferation. Except for E. brandianum, all extracts induced cellular autophagy in tumor cells with similar levels to that of rapamycin; but, only E. brandianum induced cellular apoptosis, likely through Bcl2 and BAX protein expressions. Discussion: This is the first study to report the potential antiproliferative effects of ethanol extracts of several Geraniaceae species.

Research on Mechanism of miR-214 Packaged with Lipidosome Nanoparticles on Prompting the Apoptosis of Intestinal Cancer Through Regulating p53 Pathway

Our study aimed at studying mechanism of miR-214 packaged with lipidosome nanoparticles on prompting apoptosis of intestinal cancer through regulating p53 pathway. SW480 cells were divided into blank group, empty carrier group, agonist group and group with carrier and antagonist. The negative control group was set, and groups related to p53 pathway were set as agonist group, inhibitor group and group with antagonist and inhibitor. The effect of miR-214 packaged with lipidosome nanoparticles on proliferation and apoptosis of intestinal cancer cells and p53 pathway in intestinal cancer cells was observed. Expression level of miR-214 in group with carrier and antagonist was lower than in other groups. The proportion of active cells in the group with carrier and antagonist started to be reduced notably from the second day. There was no notable declining tendency active cells’ proportion from other groups. The quantity of cell apoptosis in group with carrier and antagonist was higher than in the other groups. The expression level of cleaved Caspase-3 in the group with carrier and antagonist was notably higher than in the other groups. Moreover, expression of Bcl-2/Bax protein was reversed, while expression of p53 protein in the carrier and antagonist groups was notably higher than in the other groups. The antagonist of miR-214 packaged with lipidosome nanoparticles could target on p53 pathway. The activity of p53 pathway was reduced by miR-214, and expression of Bcl-2 was increased. The expressions levels of Bax and cleaved Caspase-3 were also reversed, and molecular mechanism was mainly related with restraining of p53 signal pathway.

Targeting Bclxl Mitigates Mcl1 Chemoresistance in Multiple Myeloma

Aim: Despite the adoption of novel therapeutic modalities, Multiple Myeloma (MM) remains incurable. The Bcl2 inhibitor Venetoclax is active in several haematologic malignancies, but the benefits in MM patients are limited to those with the t(11;14) and/or high Bcl2 expression. These results underscore the significance of Bcl2 alternative anti-apoptotic proteins (Mcl1 and BclxL) for the survival of myeloma cells. Method: We validated the anti MM effect of the Mcl1 inhibitor S63845 both in vitro utilising 11 human myeloma cell lines (HMCL) and ex vivo against n=30 primary MM tumours. Comparative analysis of RNAseq between S63845 sensitive and resistant HMCL was undertaken to identify candidate proteins that potentially modulate resistance to S63845. Treatment with S63845 and rationally selected combination partners was further evaluated in vitro, ex vivo and in vivo with flow cytometry, immunoblotting and live imaging mitochondria fitness monitoring. Results: RNAseq identified BclxL as potential mediator of resistance to S63845 in HMCL. Immunoblotting confirmed high BclxL expression and high BclxL/BclS in S63845 resistant HMCL. Five S63845 resistant HMCL (U266, ANBL6, KMS28PE, EJM, MM1R) and primary tumours were treated with S63845 combined with the BclxL inhibitor A1331852 . Combined treatment of the HMCL demonstrated a high Bliss synergy score for all the HMCL tested (54, 42, 24, 47, 45 for U266, EJM, KMS28PE, MM1R and ANBL6 respectively) and induced synergistic killing of 80% of the primary tumours treated. Dual inhibition in U266 induced an 80% drop in intracellular ATP at 4h with an increase in active Caspases 9 and 8 (4.5 and 5 fold, respectively). Similarly, the combination induced a 78% drop in mitochondrial transmembrane potential (TMRE intensity) by 4h with live imaging revealing striking mitochondrial damage as early as 40 minutes after exposure (figure). These changes were associated with a reduction of both Mcl1 and, BclxL proteins and Bim and Bid protein levels. No changes were seen in the level of Bcl2, Bak or Bax protein expression. The combination of S63845 and A1331852 in healthy NSG mice at 12.5mg/kg proved lethal due to hepatotoxicity, arguing against the clinical utility of such an approach. However, this observed anti-MM synergistic activity was recapitulated when S63845 was combined with the already approved anti-MM therapeutic panobinostat, with the induction of a significant reduction in both BclxL and Myc protein levels at 24h, and synergistic killing of 56% of primary tumours. Conclusion: High BclxL expression and BclxL/BclxS ratio correlates with resistance to the Mcl1 inhibitor S63845. A combinatorial approach targeting Mcl1 and BclxL induced immediate and significant anti-MM effect both in vitro and ex vivo but proved to be toxic in vivo. Combination of the anti-MM therapeutic panobinostat in combination with S635845 recapitulated the anti-MM activity seen with A1331852 and warrants further evaluation. Figure 1 Figure 1. Disclosures Spencer: Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria, Research Funding; Bristol Myers Squibb: Research Funding; Takeda: Honoraria, Research Funding, Speakers Bureau; STA: Honoraria.

Impact of Intravesical instillation of a Novel Biological Response Modifier (P-MAPA) on Progress of Non-Muscle Invasive Bladder Cancer Treatment in a rat model

This work describes the effects of immunotherapy with Protein Aggregate Magnesium-Ammonium Phospholinoleate-Palmitoleate Anhydride (P-MAPA) in the treatment of non-muscle invasive bladder cancer (NMIBC) in an animal model. NMIBC was induced by treating female Fischer 344 rats with N-methyl-N-nitrosourea (MNU). After treatment with MNU, the rats were distributed into four experimental groups: Control (without MNU) group, MNU (cancer) group, MNU-BCG (Bacillus Calmette-Guerin) group and MNU-P-MAPA group. P-MAPA intravesical treatment was more effective in histopathological recovery from cancer state in relation to BCG treatment. Western blot assays showed an increase in the protein levels of c-Myc, COUP-TFII and wild-type p53 in P-MAPA-treated rats in relation to BCG-treated rats. In addition, rats treated with P-MAPA intravesical immunotherapy showed the highest BAX protein levels and the lowest proliferation/apoptotic ratio in relation to BCG-treated rats, pointing out a preponderance of apoptosis. P-MAPA intravesical treatment increased the wild-type p53 levels and enhanced c-Myc/COUP-TFII-induced apoptosis mediated by p53. These alterations were fundamental for histopathological recovery from cancer and for suppress abnormal cell proliferation. This action of P-MAPA on apoptotic pathways may represent a new strategy for treating NMIBC.