Expression Of Apoptosis-associated Genes Research Articles

Interferon regulatory factor 6 (IRF6) determines intestinal epithelial cell development and immunity

Intestinal epithelial cell (IEC) responses to interferon (IFN) favor antiviral defense with minimal cytotoxicity, but IEC-specific factors that regulate these responses remain poorly understood. Interferon regulatory factors (IRFs) are a family of nine related transcription factors, and IRF6 is preferentially expressed by epithelial cells, but its roles in IEC immunity are unknown. In this study, CRISPR screens found that Irf6 deficiency enhanced IFN-stimulated antiviral responses in transformed mouse IECs but not macrophages. Furthermore, KO ofIrf6in IEC organoids resulted in profound changes to homeostasis and immunity gene expression. Irf6 KO organoids grew more slowly, and single-cell RNA sequencing indicated reduced expression of genes in epithelial differentiation and immunity pathways. IFN-stimulated gene expression was also significantly different inIrf6KO organoids, with increased expression of stress and apoptosis-associated genes. Functionally, the transcriptional changes in Irf6 KO organoids were associated with increased cytotoxicity upon IFN treatment or inflammasome activation. These data indicate a previously unappreciated role for IRF6 in IEC biology, including regulation of epithelial development and moderation of innate immune responses to minimize cytotoxicity and maintain barrier function.

The impacts of low fishmeal diet to the antioxidant capacity, endoplasmic reticulum stress, apoptosis and intestinal health of Litopenaeus vannamei as the went on of the feeding trial

Plant protein sources are the main alternative to fish meal (FM), but excessive substitution can induce inflammatory responses and oxidative stress in aquatic animals. In this study, the antioxidant capacity, endoplasmic reticulum stress (ERS), apoptosis and intestinal health of Litopenaeus vannamei at the different stages of feeding trial were evaluated after replacing FM with soy protein concentrate (SPC). Two diets were designed: HF diet (25% FM) and LF diet (15% FM + 10% soy protein concentrate), respectively. The samples were collected at 7d, 14d, 28d, 42d, 56d of the feeding trial. The results showed that the antioxidant capacity of the hepatopancreas of shrimp fed the LF diet gradually decreased after 28d. The glutathione peroxidase (GSH-PX) activity, superoxide dismutase (T-SOD) activity and total antioxidant capacity (T-AOC) showed a decreasing trend with the went on of the feeding trial and were lower than those of shrimp fed a HF diet. The length of microvilli and height of mucosal folds in shrimp fed the LF diet were significantly lower than those fed the HF diet after 42d (P<0.05), and the endoplasmic reticulum and mitochondria were swollen, and the mitochondrial matrix showed irregular structure and even almost complete dissolution at 56d. The expression of hepatopancreatic endoplasmic reticulum stress (ERS)-related genes was highest at 14d, and the hepatopancreatic endoplasmic reticulum-related genes were overall higher in shrimp fed the LF diet than those fed the HF diet after 28d. The expression of apoptosis-associated genes in hepatopancreatic cells showed an overall decreasing trend. However, the relative expression of intestinal ERS and apoptosis related gene was in the opposite trend. ERS was enhanced and apoptosis was inhibited in shrimp since 7d of the feeding trial when the dietary FM level was reduced. In conclusion, the antioxidant capacity and immune response of shrimp would be reduced as the feeding trial went on, ERS and apoptosis in the intestine of shrimp fed a LF diet would be earlier to be induced compared to those fed the HF diet.

Harmaline induces apoptosis and inhibits migration in A2780 ovarian cancer cells invitro.

Ovarian cancer is one of the most prevalent malignancies in women. Harmaline is reported to have powerful anticancer properties. We aimed to investigate the apoptotic and antimetastatic properties of harmaline in A2780 ovarian cancer cells. Cell viability, apoptosis, migration, and invasion were investigated in cells treated with harmaline. Reactive oxygen species (ROS) production, mRNA expression of apoptosis-associated genes, MMP-2, and MMP-9 were measured. Harmaline attenuated the viability of A2780 ovarian cancer cells in a dose- and time-dependent way. Furthermore, compared to NIH/3T3 mouse normal cell line (IC50 = 417 μM), the malignant A2080 cells were more sensitive to harmaline (IC50 = 300 μM after 24 h). Harmaline increased the production of ROS, raised the mRNA expression of p53 and the Bax/Bcl2 ratio. Harmaline also increased the proportion of cells in the late apoptotic and necrotic phases. MMP-2 and MMP-9's mRNA expression, gelatinase activity, and migration of A2780 cells also decreased by harmaline. These findings suggest that harmaline may have the potential to be a therapeutic drug for ovarian cancer by triggering apoptosis and suppressing invasion and migration.

Therapeutic effect of sodium alginate on bleomycin, etoposide and cisplatin (BEP)-induced reproductive toxicity by inhibiting nitro-oxidative stress, inflammation and apoptosis

Impaired spermatogenesis and male infertility are common consequences of chemotherapy drugs used in patients with testicular cancer. The present study investigated the effects of sodium alginate (NaAL) on testicular toxicity caused by bleomycin, etoposide, and cisplatin (BEP). Rats in group 1 received normal saline, while groups 2 and 3 were treated with 25 and 50 mg/kg of NaAL, respectively. Group 4 was treated with a 21-day cycle of BEP (0.5 mg/kg bleomycin, 5 mg/kg etoposide, and 1 mg/kg cisplatin), and groups 5 and 6 received BEP regimen plus 25 and 50 mg/kg of NaAL, respectively. Then, sperm parameters, testosterone levels, testicular histopathology and stereological parameters, testicular levels of malondialdehyde (MDA), nitric oxide (NO), and total antioxidant capacity (TAC), and the expression of apoptosis-associated genes including Bcl2, Bax, Caspase3, p53, and TNF-α were evaluated. Our findings revealed that NaAL improved sperm parameters, testosterone levels, histopathology, and stereology parameters in BEP-administrated rats. NaAL also improved testis antioxidant status by enhancing TAC and ameliorating MDA and NO. Further, modifications to the expression of Bcl2, Bax, Caspase3, p53, and TNF-α suggested that NaAL alleviated BEP-induced apoptosis and inflammation. Collectively, NaAL protects rats’ testes against BEP-evoked toxicity damage through the modulation of nitro-oxidative stress, apoptosis, and inflammation.

"Combination treatments of imatinib with astaxanthin and crocin efficiently ameliorate antioxidant status, inflammation and cell death progression in imatinib-resistant chronic myeloid leukemia cells".

Imatinib resistance remains a major obstacle in the treatment of chronic myelogenous leukemia (CML). Crocin (CRC) and astaxanthin (ATX) are phytochemicals with anti-cancer properties. This study aimed to explore the effects of combination treatment of Imatinib with CRC and ATX on Imatinib-resistant K562 (IR-K562) cells. After the establishment of IR-K562 cells, growth inhibitory activity was determined by the MTT assay. To test the regeneration potential, a colony formation assay was performed. Cell cycle analyses were examined by flow cytometry. Cell injury was evaluated by lactate dehydrogenase (LDH) leakage. Real-time PCR was applied to assess the expression of IL6, TNF-α, STAT3, BAD, CASP3, TP53, and Bcl-2 genes. Caspase-3 activity was determined by a colorimetric assay. Antioxidant activity was measured using a diphenylpicrylhydrazyl (DPPH) assay. After 48h of treatment, ATX (IC50 = 30µM) and CRC (IC50 = 190µM) significantly inhibited cell proliferation and colony formation ability, induced G1 cell cycle arrest and cell injury, upregulated the expression of apoptosis-associated genes, and downregulated the expression of anti-apoptotic and inflammatory genes. The combination of IM with ATX and/or CRC synergistically reduced cell viability (combination index [CI] < 1). Our data suggest that IM shows better therapeutic efficacy at lower doses when combined with ATX and/or CRC.

Kidney-tonifying formula facilitates the development and maturation of mouse preantral follicle in vitro.

Kidney-tonifying formulas are frequently used in clinical practices to enhance follicular development and maturation. This research explored the impacts of the Bushen Tiaojing formula (BSTJF) on the development of mouse preantral follicles in vitro and its relationship with granulosa cells and gonadotropins. Preantral follicles were extracted from mice and cultured with or without serum from rats that were previously treated with or without BSTJF. During cultivation, the follicles were monitored for morphological changes and developmental maturation. Exhausted medium was collected every other day for the measurement of progesterone and estradiol (E2) levels by ELISA. Granulosa cells in in-vitro medium were collected on days 8, 10, and 12 and analyzed for determining the expressions of apoptosis-associated genes (Bax, Bcl-2, and Caspase-3). Propagation and apoptosis rates of collected granulosa cells were measured by CCK-8 assay and flow cytometry. Compared with control follicles, follicles cultured with serum from BSTJF-treated rats had a higher survival rate, larger follicle diameter, higher Bcl-2 expression, and lower Bax and Caspase-3 expressions (all P ≤ 0.05). In addition, their granulosa cells presented substantially elevated proliferation (P ≤ 0.05) and a lower rate of apoptosis (P ≤ 0.05) compared with granulosa cells from control follicles. The level of E2 in the culture media of all groups increased slowly in the first 6 days. Subsequently, after formation of the antrum, the levels of E2 and progesterone were enhanced in the medium of follicles cultured with serum from BSTJF-treated rats compared with those in the media of control follicles (all P ≤ 0.05). Serum from BSTJF-treated rats facilitated the in vitro development and maturation of mouse follicles by increasing the expression of anti-apoptotic gene Bcl-2, reducing the expressions of pro-apoptotic genes Bax and Caspase-3 as well as the apoptosis of granulosa cells, promoting the proliferation of granulosa cells and increasing the secretion of E2 and progesterone in the cells.

Blockade of interleukin-6 trans-signaling prevents mitochondrial dysfunction and cellular senescence in retinal endothelial cells

Interleukin-6 (IL-6) is a multifaceted cytokine implicated in the pathogenesis of diabetic retinopathy (DR). Its activity extends through cis- and trans-signaling (TS) pathways, with cis-signaling limited to specific cell types possessing the membrane-bound IL-6 receptor, while trans-signaling broadly activates various cells without the membrane bound IL-6 receptor, including retinal endothelial cells. In this study, we determined the effects of interleukin-6 trans-signaling on mitochondrial dysfunction and cellular senescence in human retinal endothelial cells (HRECs). HRECs were cultured and treated with IL-6 + soluble IL-6R or Hyper IL-6 to activate trans-signaling, along with sgp130Fc for inhibition. RT-PCR was used to analyze gene expression changes associated with inflammation and senescence. Cellular senescence was assessed using SA β-gal staining. Mitochondrial function was evaluated using Seahorse XFe24 Bioanalyzer. IL-6 trans-signaling induced inflammatory gene expression as indicated by the upregulation of ICAM1, MCP1, and SERPINA3 levels. Additionally, it reduced mitochondrial respiration and oxidative phosphorylation, and these effects were counteracted by sgp130Fc. Moreover, IL-6 trans-signaling led to altered expression of apoptosis-associated genes, including downregulation of FIS1, BCL2, and MCL1, while promoting cellular senescence, a phenomenon mitigated by sgp130Fc. These results not only deepen our understanding of IL-6 in DR but also carry broader implications for age-related diseases and the aging process itself. This study underscores the potential therapeutic value of targeting IL-6 trans-signaling with sgp130Fc as a promising anti-inflammatory approach for DR and potentially other inflammatory conditions. Further in-vivo investigations are warranted to elucidate the function of IL-6 trans-signaling in aging-related pathologies and overall organismal health.

Silencing suppressor of cytokine signaling 3 induces apoptosis and activates the p-STAT3/NF-κB pathway in hypoxic cultivated H9c2 cells.

Suppressor of cytokine signaling 3 (SOCS3) plays a significant role in the process of myocardial adaptation to chronic hypoxia. SOCS3 finely regulates cell signaling cross-talk that occurs between NF-κB and STAT3 during the compensatory protective response. However, the role and mechanism of SOCS3 in hypoxic cardiomyocytes are not fully understood. In the study, we investigated the effect of SOCS3 on the p65 and STAT3 signaling pathways and further examined the potential molecular mechanism involved in regulating apoptosis. Our data showed that SOCS3 silencing could upregulate Ac-p65, p-p65, and p-STAT3 expression in nuclear extracts of H9c2 cells that received hypoxic treatment for 24, 48, and 72 h. SOCS3 silencing also remarkably increased the DNA-binding activity of the p65 motif in hypoxic cultivated H9c2 cells. We also found that SOCS3 knockdown increased cleaved-caspase-3, Bax, and PUMA expression and decreased cleaved PARP and Bcl-2 in expression in hypoxic H9c2 cells. Silencing of SOCS3 caused an increase in LDH leakage from injured cardiomyocytes and reduced cell viability under conditions of hypoxic stress. Furthermore, SOCS3 silencing enhanced the apoptosis of H9c2 cells at 72 h of hypoxia. These findings suggest that knockdown of SOCS3 leads to excessive activation of the NF-κB pathway, which, in turn, might promote apoptosis under conditions of chronic hypoxia.

In vitro effects of sodium butyrate, a histone deacetylase inhibitor on canine mammary tumour subtypes

AbstractMalignant canine mammary tumours (CMTs) are subdivided as carcinomas (C), sarcomas (S) and carcinosarcomas (CS) according to their histological type. Due to the varying responses to different treatments, novel therapeutic approaches are required. Sodium butyrate (NaBu) is the first histone deacetylase inhibitor and exhibits anti-cancer activity in different types of human cancers. However, no study is available in the literature evaluating the effects of NaBu on CMT cells. Therefore, for the first time, we aimed to investigate the in vitro efficacy of NaBu on three subtypes of CMT cells. Primary cells were isolated from neoplastic tissues of three dogs with CMT. Cell viability, apoptotic percentage and changes in the cell morphology, apoptosis-associated gene and protein expression levels were evaluated. Our findings showed that NaBu decreased the growth of the cells and increased the percentage of apoptosis in three subtypes of CMT cells. Additionally, NaBu treatment affected the expression levels of genes and proteins associated with apoptosis, especially in S cells. Our preliminary results, for the first time, revealed the efficacy of NaBu in CMT cells and the importance of the histological subtype in NaBu response. However, the molecular classification of CMT and the underlying mechanisms of the different NaBu responses should be further investigated.

Use of multi-walled carbon nanotubes (MWCNTs) stabilized in Arabic gum colloidal solution to induce genotoxicity and apoptosis of human breast and lung cancer cell lines

Introduction: Increased mortality owing to breast and lung cancer is an urgent global medical issue. Nanotherapy is one of the most widely used and recent procedures for combating cancer. Therefore, this study aimed to assess the beneficial effects of multi-walled carbon nanotubes (MWCNTs) to precisely target breast (MCF-7) and lung cancer (A549) cell lines. Multi-walled carbon nanomaterials are considered as a typical biocompatible nanomaterial that can be used in drug delivery without causing side effects. On the contrary, the metallic nanoparticles, even that of noble metals, are reported to have side effects that are still unavoidable.Methods: The MWCNTs were purified and stabilized using Arabic gum in an aqueous solution under ultrasonic radiation. Scanning and transmission electron microscopy (SEM and TEM, respectively) were employed to characterize the morphology of the MWCNTs, and elemental analysis was performed using SEM to determine their chemical composition.Results: The investigated MWCNTs had an average diameter of 10–12 nm and were mainly composed of 99.4% carbon and other trace elements. The Arabic gum emulsifying agent was found a powerful stabilizing agent for the investigated MWCNTs in aqueous solutions. The cytotoxic effect of the MWCNT/ Arabic gum colloidal solution on MCF-7 and A549 was evaluated by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays, and the expression of apoptosis-related genes was assessed using quantitative real-time polymerase chain reaction and a human apoptosis signaling antibody array. This analysis aimed to clarify additional pathways involved, including those mediated by interleukin-1, tumor necrosis factor-α, and ceramide signaling pathways enrichments. Concerning the inhibition of cell growth, the efficacy of MWCNT/Arabic gum IC50 of MCF-7 (48.23 μg/mL) was higher than IC50 of A549 (100.1 μg/mL). MWCNT/Arabic gum enhanced the mRNA expression of apoptosis-associated genes encoding P53, Bax, and Caspases (9, 8, and 3). Upregulation of the apoptotic signaling pathway in the MCF-7 cell lysates was observed before and after exposure to the MWCNT/Arabic gum colloidal solution.Conclusion: The physicochemical properties of MWCNT/Arabic gum exhibited remarkable apoptotic activity in breast and lung cancer cell lines. However, further in vivo investigations are recommended to clarify the other molecular aspects related to the use of MWCNT/Arabic gum.

Orotic acid induces apoptotic death in ovarian adult granulosa tumour cells and increases mitochondrial activity in normal ovarian granulosa cells

Orotic acid (OA) is a natural product that acts as a precursor in the pyrimidine nucleotide biosynthesis pathway. Most studies concerning administration of OA focus on its therapeutic effects; however, its effect on tumours is unclear. We aimed to determine whether treatment with OA influences the viability and apoptosis of normal (HGrC1) and tumour-derived (KGN) human ovarian granulosa cells. The effects of OA (10–250 μM) on viability and apoptosis of both cell lines were determined by using alamarBlue and assessing caspase-3/7 activity, respectively. Annexin V binding and loss of membrane integrity were evaluated in KGN cells. The cell cycle and proliferation of HGrC1 cells were assessed by performing flow cytometric and DNA content analyses, respectively. The influence of OA (10 and 100 μM) on cell cycle- and apoptosis-related gene expression was assessed by RT-qPCR in both cell lines. Mitochondrial activity was analysed by JC-1 staining in HGrC1 cells. In KGN cells, OA reduced viability and increased caspase-3/7 activity, but did not affect mRNA expression of Caspase 3, BAX, and BCL2. OA enhanced proliferation and mitochondrial activity in HGrC1 cells without activating apoptosis. This study demonstrates that the anti-cancer properties of OA in ovarian granulosa tumour cells are not related to changes in apoptosis-associated gene expression, but to increased caspase-3/7 activity. Thus, OA is a promising therapeutic agent for ovarian granulosa tumours. Further, our results suggest that differences in basal expression of cell cycle- and apoptosis-related genes between the two cell lines are responsible for their different responses to OA.

Exogenous Hydrogen Sulfide Induces A375 Melanoma Cell Apoptosis Through Overactivation of the Unfolded Protein Response.

Melanomas are highly malignant and rapidly develop drug resistance due to dysregulated apoptosis. Therefore, pro-apoptotic agents could be effective for the management of melanoma. Hydrogen sulfide is ubiquitous in the body, and exogenous hydrogen sulfide has been reported to show inhibitory and pro-apoptotic effects on cancer cells. However, whether high concentrations of exogenous hydrogen sulfide have pro-apoptotic effects on melanoma and its mechanisms remain unknown. Hence, this study aimed to explore the pro-apoptotic effects and mechanisms of exogenous hydrogen sulfide on the A375 melanoma cell line treated with a hydrogen sulfide donor (NaHS). The cell proliferation test, flow cytometric analysis, Hoechst 33258 staining, and Western blotting of B-cell lymphoma 2 and cleaved caspase-3 were used to explore the pro-apoptotic effects of hydrogen sulfide on A375 cells. The transcriptional profile of NaHS-treated A375 cells was further explored via high-throughput sequencing. Western blotting of phosphorylated inositol-requiring enzyme 1α (p-IRE1α), phosphorylated protein kinase R-like ER kinase (p-PERK), phosphorylated eukaryotic translation initiation factor 2α (p-eIF2α), C/EBP homologous protein, glucose-regulating protein 78, IRE1α, PERK, and eIF2α was performed to verify the changes in the transcriptional profile. NaHS inhibited A375 melanoma cell proliferation and induced apoptosis. The endoplasmic reticulum stress unfolded protein response and apoptosis-associated gene expression was upregulated in NaHS-treated A375 melanoma cells. The overactivation of the unfolded protein response and increase in endoplasmic reticulum stress was verified at the protein level. Treatment with NaHS increased endoplasmic reticulum stress, which triggered the overactivation of the unfolded protein response and ultimately lead to melanoma cell apoptosis. The pro-apoptotic effect of NaHS suggests that it can be explored as a potential therapeutic agent in melanoma.

Single Fraction and Hypofractionated Radiation Cause Cochlear Damage, Hearing Loss, and Reduced Viability of Merlin-Deficient Schwann Cells.

Vestibular schwannomas (VS) are benign intracranial tumors caused by loss of function of the merlin tumor suppressor. We tested three hypotheses related to radiation, hearing loss (HL), and VS cell survival: (1) radiation causes HL by injuring auditory hair cells (AHC), (2) fractionation reduces radiation-induced HL, and (3) single fraction and equivalent appropriately dosed multi-fractions are equally effective at controlling VS growth. We investigated the effects of single fraction and hypofractionated radiation on hearing thresholds in rats, cell death pathways in rat cochleae, and viability of human merlin-deficient Schwann cells (MD-SC). Adult rats received cochlear irradiation with single fraction (0 to 18 Gray [Gy]) or hypofractionated radiation. Auditory brainstem response (ABR) testing was performed for 24 weeks. AHC viabilities were determined using immunohistochemistry. Neonatal rat cochleae were harvested after irradiation, and gene- and cell-based assays were conducted. MD-SCs were irradiated, and viability assays and immunofluorescence for DNA damage and cell cycle markers were performed. Radiation caused dose-dependent and progressive HL in rats and AHC losses by promoting expression of apoptosis-associated genes and proteins. When compared to 12 Gy single fraction, hypofractionation caused smaller ABR threshold and pure tone average shifts and was more effective at reducing MD-SC viability. Investigations into the mechanisms of radiation ototoxicity and VS radiobiology will help determine optimal radiation regimens and identify potential therapies to mitigate radiation-induced HL and improve VS tumor control.

Emamectin benzoate exposure impaired porcine oocyte maturation

Emamectin benzoate (EB) is a widely used insecticide that can damage the central nervous and immune systems. EB exposure significantly reduced the number of eggs laid, hatching rate, and developmental rate of lower organisms such as nematodes. However, effects of EB exposure on the maturation of higher animals such as porcine oocytes remains unknown. Here we reported that EB exposure severely impaired porcine oocyte maturation. EB exposure with 200 μM prevented cumulus expansion and reduced the rates of first polar body (pb1) extrusion, cleavage and blastocyst after parthenogenetic activation. Moreover, EB exposure disrupted spindle organization, chromosome alignment, and polymerization of microfilaments, but also apparently decreased the levels of acetylated α-tubulin (Ac-Tub) in oocytes. In addition, EB exposure perturbed mitochondria distribution and increased levels of reactive oxygen species (ROS), but did not affect the distribution of cortical granules (CGs) in oocytes. Excessive ROS caused DNA damage accumulation and induced early apoptosis of oocytes. EB exposure led to the abnormal expression of cumulus expansion and apoptosis-associated genes. Altogether, these results demonstrate that EB exposure impaired nuclear and cytoplasmic maturation of porcine oocytes probably through oxidative stress and early apoptosis.

Enhanced anti-cancer effect of artemisinin- and curcumin-loaded niosomal nanoparticles against human colon cancer cells.

Colorectal cancer (CRC) is the third broadly identified cancer in the world. The ineffectiveness of colorectal cancer treatment is redundantly reported. Natural bioactive compounds have gained popularity in reducing the drawback of conventional anti-cancer agents. Curcumin (Cur) and Artemisinin (Art) are materials of a natural source that have been utilized to treat numerous kinds of cancers. Although the benefits of bioactive materials, their utilization is limited because of poor solubility, bioavailability, and low dispersion rate in aqueous media. Nano delivery system such as niosome can improve the bioavailability and stability of bioactive compounds within the drug. In current work, we used Cur-Art co-loaded niosomal nanoparticles (Cur-Art NioNPs) as an anti-tumor factor versus colorectal cancer cell line. The synthesized formulations were characterized using dynamic light scattering, scanning electron microscopy, and FTIR. The proliferation ability of the cells and expression of apoptosis-associated gene were MTT assay and qRT-PCR, respectively. Cur-Art NioNPs exhibited well distributed with an encapsulation efficiency of 80.27% and 85.5% for Cur and Art. The NioNPs had good release and degradation properties, and had no negative effect on the survival and proliferation ability of SW480 cells. Importantly, nanoformulation form of Cur and Art significantly displayed higher toxicity effect against SW480 cells. Furthermore, Cur-Art NioNPs increased Bax, Fas, and p53 gene expressions and suppressed Bcl2, Rb, and Cyclin D 1 gene expressions. In summary, these results display the niosome NPs as a first report of nano-combinational application of the natural herbal substances with a one-step fabricated co-delivery system for effective colorectal cancer.

Harmaline exerts potentially anti-cancer effects on U-87 human malignant glioblastoma cells in vitro

Harmaline is a β-carboline alkaloid that can be extracted from the seeds of Peganum harmala. Harmaline has been shown to exhibit a potent cytotoxic effect against tumor cells. In this study, the anti-glioblastoma activity of harmaline was investigated in vitro. Cell viability, apoptosis, and cell cycle arrest were assessed in U-87 cells treated with harmaline at different doses. Reactive oxygen species (ROS) generation and the mRNA expression of apoptosis-associated genes were assessed. The anti-metastatic effect of harmaline on U-87 cells was evaluated by gelatin zymography assay where matrix metalloproteinase [MMP]-2/-9 enzymatic activity was measured, and the scratch assay was used to assess migratory responses. Flow cytometry demonstrated that harmaline could suppress the proliferation and induce sub-G1 cell cycle arrest and apoptotic cell death in glioblastoma cells. Harmaline treatment was also associated with an upregulation of the cell cycle-related genes, p21 and p53, and pro-apoptotic Bax, as well as the induction of ROS. The zymography assay indicated that the essential steps of metastasis were potently suppressed by harmaline through inhibiting the expression of MMP-2 and - 9. In addition, the migration of U-87 cells was significantly reduced after harmaline treatment. Our data suggest a basis for further research of harmaline which has potential cytotoxic activities in glioblastoma cells; inducing cell cycle arrest and apoptosis, repression of migration, possibly invasion, and metastasis.

Influence of fabrication method on the biological properties of modified PEEK.

To investigate the influence of fabrication method on the biological properties of modified PEEK. Two materials were tested in this study: press and CAD/CAM milled ceramic-reinforced PEEK (BioHPP, Bredent, Germany). The residual diphenyl sulfone in these two materials was detected and their biological effects on human gingival fibroblasts (HGFs) were evaluated by assaying cell viability, apoptosis, and inflammation. Diphenyl sulfone was not detected in press and milled BioHPP. The cell viability of them complied with ISO standards. The mRNA expression levels of Bax and Caspase-3 were up-regulated and that of Bcl-2 was downregulated when HGFs were cultured with the eluates of these two materials. There was no difference in cell viability, apoptosis, and inflammation between them. Hence, the fabrication method did not affect the biological properties of modified PEEK, the press and CAD/CAM milled modified PEEK all presented favorable cytocompatibility despite the expression of apoptosis-associated genes at the mRNA level.

Abstract IA027: Role of stress-survival pathways and transcriptomic alterations in progression of colorectal cancer: A Hispanic health disparities perspective

Abstract Essential to improving treatment, prognosis, and detection strategies are the identification and validation of new ethnicity-specific genomic profiles in cellular stress and survival pathways as effective predictors of colorectal cancer (CRC). These pathways are increasingly implicated in tumorigenesis of CRC and in providing resistance to chemotherapy-induced cell death. In this study, we have explored the role of these pathways in the biological significance of the expression of stress-survival pathway-associated genes in CRC progression and their role in promoting tumor aggressiveness from the Hispanic health disparities perspective. Transcript expression of oxidative stress and apoptosis-associated gene was evaluated in CRC gene microarray and RNAseq datasets to obtain 28 target genes. These genes were screened for transcript-level expression in six CRC cell lines and cDNA arrays containing the tumor (n=40) from different stages, and control (n=8) samples. The protein expression was evaluated by immunohistochemistry (IHC) in CRC microarrays (TMAs) containing different stages of tumor (n = 108) and control (n = 12) tissues. Target genes were also analyzed in Hispanic and Non-Hispanic White (NHWs) tissues at the transcript level. The analysis of target genes FOXM1, CDK1, CDK4, Chek1, and MCM10 showed consistent upregulation in CRC cell lines, cDNA arrays, and TMAs. The differential expression was also associated with the tumor stages. Among the target genes, Chek1, MCM10, and FOXM1 were found to be differentially expressed in Hispanic tissues when compared to the NHW samples. No significant difference was observed in the expression levels of CDK1 and CDK4 between the Hispanic and NHW tissues. Eight genes that did not show differential expression in CRC cell lines, were also seen to be upregulated in the Hispanic tissues, suggesting the influence of ethnicity-specific genetic makeup on the expression of these genes. The genes identified to be upregulated in Hispanic tissues may be used as potential biomarkers or as therapeutic targets in the Hispanic population. Citation Format: Aditi Kulkarni, Urbashi Basnet, Frances Rangel, Sourav Roy. Role of stress-survival pathways and transcriptomic alterations in progression of colorectal cancer: A Hispanic health disparities perspective [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr IA027.

Preparation of chitosan modified poly(lactic-co-glycolic acid) nanoparticles containing hawthorn proanthocyanidins: SCC25 cell cycle arrest, antiproliferation, and apoptosis studies

The present study aims at the preparation of a chitosan-PLGA-based proanthocyanidin from hawthorn fruit loaded nano-system (CS-PLGA-PHL) and investigate its inhibitory effect on SCC-25 cells and the related mechanisms. Methods: CS-PLGA-PHL nanocomposites were constructed by emulsion-solvent evaporation. The effect of CS-PLGA-PHL on the proliferation of human normal squamous epithelial hNOK cells were detected. The antitumor activity of CS-PLGA-PHL on SCC-25 cells were studied by cytotoxicity test, cell cycle and apoptosis analysis. The apoptotic protein and gene expression in SCC-25 cells was detected by western blotting and q-PCR. Results: The particle size distribution of CS-PLGA-PHL was narrow with an average of 177.60±41.00 nm. CS-PLGA-PHL did not show significant cytotoxicity to hNOK cells (IC50 &gt; 50 μg/mL), with strong cytotoxicity to SCC-25 cells (IC50 = 1.21 μg/mL), which could block the G0/G1 phase of SCC-25 cells and increase the distribution of G2/M phase. Meanwhile, the apoptosis induction of CS-PLGA-PHL was higher than that of free PHL and PLGA-PHL group (P &lt;0.05). The cell cycle inhibition and apoptosis induction may be due to the up regulation of apoptosis-associated proteins and genes. Conclusion: CS-PLGA-PHL nanocomposites can inhibit the cell cycle and promote apoptosis of SCC-25 cells by up regulating the expression of apoptosis-associated proteins and genes, suggesting that it has potential as a chemotherapeutic drug for the treatment of oral squamous cell carcinoma.

A simple, robust and scalable route to prepare sub-50 nm soft PDMS nanoparticles for intracellular delivery of anticancer drugs

Soft nanoparticles (NPs) have recently emerged as a promising material for intracellular drug delivery. In this regard, NPs derived from polydimethylsiloxane (PDMS), an FDA approved polymer can be a suitable alternative to conventional soft NPs due to their intrinsic organelle targeting ability. However, the available synthesis methods of PDMS NPs are complicated or require inorganic fillers, forming composite NPs and compromising their native softness. Herein, for the first time, we present a simple, robust and scalable strategy for preparation of virgin sub-50 nm PDMS NPs at room temperature. The NPs are soft in nature, hydrophobic and about 30 nm in size. They are stable in physiological medium for two months and biocompatible. The NPs have been successful in delivering anticancer drug doxorubicin to mitochondria and nucleus of cervical and breast cancer cells with more than four-fold decrease in IC50 value of doxorubicin as compared to its free form. Furthermore, evaluation of cytotoxicity in reactive oxygen species detection, DNA fragmentation, apoptosis-associated gene expression and tumor spheroid growth inhibition demonstrate the PDMS NPs to be an excellent candidate for delivery of anticancer drugs in mitochondria and nucleus of cancer cells.