Cellular Proliferation Assays Research Articles

Abstract PO5-24-08: Differential exploitation of Cation Transport Regulator homolog 1 (CHAC1) by Wild-type and mutant p53

Abstract Background: Cancer cells are constantly exposed to harsh conditions like nutrient deprivation, hypoxia, and calcium imbalance leading to the induction of UPR. In its early phase, UPR aims to restore homeostasis while prolonged stress leads to induction of apoptosis. Thus, it is not surprising to observe that expression of the UPR components has been reported to both promote as well as suppress tumorigenesis. Studies have shown that Cation transport regulator homolog 1 (CHAC1) plays a dual role in cancer progression. The objective of the present study is to investigate the p53 mediated regulation of CHAC1 in breast cancer. Methods: We initially subjected a breast cancer tissue microarray (TMA) to immunohistochemistry (IHC) with p53 and CHAC1 specific antibody. We further did in vitro experiments like western blotting, qRT-PCR to see the expression of CHAC1 when wild type (WT) p53 was either induced using camptothecin (CPT) and Nutlin-3 in WT p53 expressing breast cancer cells or by the ectopic WT and mutant p53 transfection in null p53 breast cancer cells (MDA-MB-157). The Chromatin immunoprecipitation (ChIP) assay was done to find a potential binding site of p53 on CHAC1 promoter. Further, in vitro study was done to elucidate a potential role of CHAC1 in cell proliferation. Results: The IHC staining analysis revealed that there was no significant difference in the CHAC1 expression between high and low p53 staining tissue cores as high CHAC1 staining was observed in both cases. Further, in vitro analysis reinforced the findings of CHAC1 in TMAs as cell line studies showed that both forms of p53 had enhancing effect on CHAC1 levels. Upon induction of WT p53 and/or ectopic transfection of WT and mutant p53, we observed that CHAC1 levels were upregulated in a dose dependent manner. ChIP assay revealed that while WT p53 occupies the promoter region of the CHAC1 gene to induce its expression, mutant p53 may be inducing CHAC1 expression through an unknown mediator. These observations provide the basis that CHAC1 may be a novel transcriptional target of p53. Ectopic transfection of WT and mutant 53 showed opposite effect on cellular proliferation as evident from BrdU cellular proliferation assay in MDA-MB-157 cells. Further, we showed that CHAC1 enhanced breast cancer cell proliferation possibly via upregulation of PI3K/Akt/mTOR pathway and may likely be a player in angiogenesis as shown by in vitro angiogenesis assay. Conclusion: Taken together, the data from present study suggests that the effect of CHAC1 on cellular proliferation was associated with the status of p53 in breast cancer cells. The results suggest a plausible explanation for the differential behavior of CHAC1 in tumorigenesis. Citation Format: Vikrant Mehta, Harish Chander. Differential exploitation of Cation Transport Regulator homolog 1 (CHAC1) by Wild-type and mutant p53 [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-08.

Boswellic acid as a potential adjunct for bone healing after endodontic surgery: In vitro study

Abstract Introduction: The role of Acetyl -11-keto-β-boswellic acid (AKBA) in regulating osteoblast differentiation was recently brought to light. Therefore, the current study was designed to explore the osteogenic differentiation capability of AKBA on bone marrow mesenchymal stem cells (BMMSCs) as a potential therapeutic agent to accelerate the healing process in apicoectomy. Materials and Methods: BMMSCs were characterized by flow cytometry. Cellular viability and proliferation assays were used with different concentrations of AKBA. Cells were divided into 5 groups to test osteogenic differentiation: Group I: negative control, Group II: positive control, Group III: BMMSCs were treated with 1 μM AKBA, Group IV: BMMSCs were treated with 0.1 μM AKBA, and Group V: BMMSCs were treated with 0.01 μM AKBA. Mineralization assays and gene expression analysis were assessed, and the significance difference between groups was established at P < 0.05. Results: The flow cytometry analysis demonstrated that BMMSCs had positive expression for mesenchymal stem cell marker and negative expression for hematopoietic markers. The concentration of 0.01 μM gave significantly higher cell density than the untreated cells after 7 days (P < 0.05). Cells treated with 0.1 and 0.01 μM AKBA revealed a significantly higher ALP activity, alizarin red, and von Kossa staining than control groups (P < 0.05). High expression of osteogenic genes was detected in BMMSCs treated with 0.1 μM AKBA (P < 0.05). Conclusions: It was declared that the concentration of 0.1 μM AKBA has no toxicity on BMMSC viability and proliferation with an impact on BMMSC osteogenic differentiation. Therefore, AKBA (0.01 μM) could be used in bone regeneration during periradicular surgery.

Novel combinatorial autophagy inhibition therapy for triple negative breast cancers

BackgroundTriple negative breast cancer (TNBC) has the worst prognosis among breast cancer subtypes. It is characterized by lack of estrogen, progesterone and human epidermal growth factor 2 receptors, and thus, have limited therapeutic options. Autophagy has been found to be correlated with poor prognosis and aggressive behaviour in TNBC. This study aimed to target autophagy in TNBC via a novel approach to inhibit TNBC progression. MethodsImmunoblotting and confocal microscopy were carried out to examine the effect of tumor microenvironmental stressors on autophagy. Cellular proliferation and migration assays were used to test the effect of different autophagy inhibitors and standard chemotherapy alone or in combination. In vivo xenograft mouse model was utilized to assess the effect of autophagy inhibitors alone or in combination with Paclitaxel. High resolution mass spectrometry based proteomic analysis was performed to explore the mechanisms behind chemoresistance in TNBC. Lastly, immunohistochemistry was done to assess the correlation between autophagy related proteins and clinical characteristics in TNBC tissue specimens. ResultsMetabolic stressors were found to induce autophagy in TNBC cell lines. Autophagy initiation inhibitors, SAR405 and MRT68921, showed marked synergy in their anti-proliferative activity in both chemosensitive and chemoresistant TNBC cell models. Paradoxically, positive expression of autophagosome marker LC3 was shown to be associated with better overall survival of TNBC patients. ConclusionIn this study, a novel combination between different autophagy inhibitors was identified which inhibited tumor cell proliferation in both chemosensitive and chemoresistant TNBC cells and could result in development of a novel treatment modality against TNBC.

Abstract LB033: Identification of small molecule Pan-TEAD inhibitors disrupting YAP-TEAD protein-protein interaction and targeting gastric cancer cells

Abstract Because of the Hippo pathway’s unique ability to restrict TAZ/YAP-TEAD in promoting regeneration, any abnormality of its core components, may promote the migration, invasion, and malignancy of cancer cells. Aberrant overexpression of YAP/TAZ-TEAD promotes tumorigenesis and is therefore considered oncogenes in many solid tumors. Drug resistance is a major factor undermining the efficacy of cancer drugs. As YAP/TAZ-TEAD complex plays a role in intrinsic and acquired resistance to various chemotherapeutic and targeted therapy drugs, there is increasing interest in combining a TEAD inhibitor with various cancer therapies. Since elevated YAP/TAZ-TEAD activity has been implicated in multiple cancer types at various stages of cancer progression and cancer types, Drugging the TEAD hydrophobic pocket, which was discovered in 2015, remains as an attractive and proven strategy to modulate the activity. Despite the progresses, only 3 small molecule TEAD inhibitors are currently being tested in Phase I clinical trials which includes: VT3989b NCT04665206 from Vivacare, IK-930b NCT05228015 from Ikena Oncology and IAG-933, NCT0485737 from the Novartis Oncology. As such, there is a need to uncover alternative TEAD inhibitors for cancer therapeutics. We report novel small molecule inhibitors that form a covalent complex with the conserved cysteine of the TEAD palmitoylation site. Compounds inhibited TEAD transcriptional target genes in cancer cells. Cellular proliferation and colony forming assay data strongly displayed cellular sensitivity in Hippo signaling altered cancer cell lines. TEAD1 dependency of gastric cancer cell lines enhance cellular sensitivity in response to small molecule inhibitors. Transcriptional profiling and RNA seq data indicate that the covalent inhibitors specifically downregulate down-stream transcriptional target genes. With our exciting key data, we are exploring multiple other oncology targets to use our Pan-TEAD inhibitors either as a monotherapy or combination therapy. We expect to complete our Technology Disclosure on additional targets before the meeting and to add exciting data for the Cancer drug development community. Citation Format: Ramesh Kumar, Joel Toh, Joanne Thian, Noorul Farzana Mohideen, Jialin Sun, Sayan Chakraborty, Jayantha Gunaratne, Wanjin Hong. Identification of small molecule Pan-TEAD inhibitors disrupting YAP-TEAD protein-protein interaction and targeting gastric cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB033.

Inhibition of autophagy initiation: A novel strategy for oral squamous cell carcinomas

BackgroundOral squamous cell carcinoma (OSCC) is one of the most common forms of oral cancer and is known to have poor prognostic outcomes. Autophagy is known to be associated with aggressive tumor biology of OSCC. Hence, this study aimed to develop a novel therapeutic strategy against OSCC by targeting the autophagic pathway. MethodsImmunoblotting, and confocal microscopy were used to examine the effect of tumor microenvironmental stressors on the autophagy activity. Cellular proliferation and migration assays were performed to assess the anti-cancer activity of standard chemotherapy and autophagy initiation inhibitors, either alone or in combination. High resolution mass-spectrometry based proteomic analysis was utilized to understand the mechanisms behind chemoresistance in OSCC models. Finally, immunohistochemistry was performed to determine associations between autophagy markers and clinicopathological characteristics. ResultsTumor microenvironmental stressors were shown to induce autophagy activity in OSCC cell lines. Novel combinations of chemotherapy and autophagy inhibitors as well as different classes of autophagy inhibitors were identified. Combination of MRT68921 and SAR405 demonstrated marked synergy in their anti-proliferative activity and also showed synergy with chemotherapy in chemoresistant OSCC cell models. Autophagy was identified as one of the key pathways involved in mediating chemoresistance in OSCC. Furthermore, TGM2 was identified as a key upstream regulator of chemoresistance in OSCC models. Finally, positive staining for autophagosome marker LC3 was shown to be associated with low histological grade OSCC. ConclusionIn conclusion, this study identified a combination of novel autophagy inhibitors which can potently inhibit proliferation of both chemosensitive as well as chemoresistant OSCC cells and could be developed as a novel therapy against advanced OSCC tumors.

Hyperthermia inhibits cellular function and induces immunogenic cell death in renal cell carcinoma

BackgroundIn recent years, hyperthermia has been widely applied as a novel strategy for cancer treatment due to its multiple antitumour effects. In particular, the potential influences of hyperthermia on the tumour immune microenvironment may improve the efficacy of immunotherapies. However, the effect of hyperthermia on renal cell carcinoma (RCC) has not been well characterized until now.MethodsIn the present study, we primarily evaluated the effects of hyperthermia on cellular function via cellular proliferation, migration, invasion and apoptosis assays. In addition, the influence of hyperthermia on the immunogenicity of RCC cells was analysed using flow cytometry analysis, enzyme-linked immunosorbent assays, and immunofluorescent (IF) staining.ResultsOur results demonstrate that hyperthermia significantly inhibits RCC cell proliferation, migration, and invasion and promotes cell apoptosis. In addition, we verified that hyperthermia improves the immunogenicity of RCC cells by inducing immunogenic cell death.ConclusionOur findings suggest that hyperthermia is a promising therapeutic strategy for RCC.

The effect of augmenting suture material with magnesium and platelet-rich plasma on the in vitro adhesion and proliferation potential of subacromial bursa-derived progenitor cells

Connective tissue subacromial bursa-derived progenitor cells (SBDCs) have been suggested as a potent biologic augment to promote healing of the repaired rotator cuff tendon. Maximizing the amount of retained progenitor cells at the tendon repair site is essential for ensuring an optimal healing environment, warranting a search for proadhesive and proliferative adjuvants. The purpose was to evaluate the effect of magnesium (Mg), platelet-rich plasma (PRP), and a combination of both adjuvants on the invitro cellular adhesion and proliferation potential of SBDCs on suture material commonly used in rotator cuff surgery. SBDCs were isolated from subacromial bursa samples harvested during rotator cuff repair and cultured in growth media. Commercially available collagen-coated nonabsorbable flat-braided suture was cut into 1-inch pieces, placed into 48-well culture dishes, and sterilized under ultraviolet light. Either a one-time dose of 5 mM sterile Mg, 0.2 mL of PRP, or a combination of both adjuvants was added, while a group without treatment served as a negative control. Cellular proliferation and adhesion assays on suture material were performed for each treatment condition. Augmenting the suture with Mg resulted in a significantly increased cellular adhesion (total number of attached cells) of SBDCs compared to PRP alone (31,527±19,884 vs. 13,619±8808; P<.001), no treatment (31,527±19,884 vs. 21,643±8194; P=.016), and combination of both adjuvants (31,527±19,884 vs. 17,121±11,935; P<.001). Further, augmentation with Mg achieved a significant increase in cellular proliferation (absorbance) of SBDCs on suture material when compared to the PRP (0.516±0.207 vs. 0.424±0.131; P=.001) and no treatment (0.516±0.207 vs. 0.383±0.094; P<.001) group. The combination of Mg and PRP showed a significantly higher proliferation potential compared to PRP alone (0.512±0.194 vs. 0.424±0.131; P=.001) and no treatment (0.512±0.194 vs. 0.383±0.094; P<.001). There were no significant differences in the remaining intergroup comparisons (P>.05, respectively). Augmenting suture material with Mg resulted in a significantly increased cellular adhesion of SBDCs compared to untreated suture material, as well as augmentation with PRP alone or a combination of both adjuvants. Further, Mg with or without PRP augmentation achieved a significant increase in the cellular proliferation of SBDCs on suture material compared to untreated sutures and augmentation with PRP alone. Application of Mg may be a clinically feasible approach to optimizing the use of SBDCs as a biological augment in rotator cuff repair, while combined augmentation with PRP may harness the full potential for optimized tissue recovery due to the high concentration of PRP-derived growth factors.

Preparation of chitosan‐embedded recombinant human epidermal growth factor nanoparticles as accelerating compounds for skin remodelling in chronic lesions

AbstractThe chronic lesion has become a major biological difficulty. Using nanoparticles as drug delivery systems is remarkable nowadays. The unique properties of chitosan in combination with epidermal growth factor (EGF) can accelerate the wound‐healing process.In this study, Chitosan‐EGF (CS‐EGF) nanoparticles were manipulated and characterized by Transmission Electron Microscopy (TEM) microscopy, dynamic light scattering (DLS) method, and Fourier Transform Infrared Spectroscopy (FTIR) radiation. The antibacterial effect was estimated by minimum inhibitory concentration (MIC)/minimum bactericidal concentration (MBC) methods and the proliferation assay was measured on the HFF‐1 (human fibroblast cell line). Then, migration assay was accomplished and the gene expression analysis for transforming growth factor‐beta (TGF‐β), vascular endothelial growth factor (VEGF), and platelets‐derived growth factor (PDGF) was manipulated by the real‐time‐PCR method. The obtained results were considered statistically significant with P &lt; 0.05.Obtained results illustrated no toxic effect on the HFF‐1 cell line treated with CS‐EGF. In cellular proliferation and migration assays, CS‐EGF nanoparticles demonstrated 2‐folds higher than the control. In the duration of 72 h of the experiment, and concentration of 10 µM, 90 ±10% of cells were migrated and the whole scratch was covered by fibroblasts. The real‐time‐PCR analysis also showed 7.5, 4.5‐, and 7‐fold upregulation of all TGF‐β, VEGF, and PDGF genes in comparison with the control group.This study confirmed that using chitosan nanoparticles as a carrier for EGF can accelerate cellular remodelling and annihilate bacterial infection in the process of treatment. As CS‐EGF nanoparticles in the acceleration of the skin remodelling process showed promising results, subsequent studies might be useful.

Hypericum perforatum Oil and Vitamin A Palmitate-Loaded Gelatin Nanofibers Cross-Linked by Tannic Acid as Wound Dressings.

Recent studies in wound dressing applications offer new therapies to promote the wound healing process. The main strategy of this study is to combine the traditional perspective of using medicinal oils with polymeric scaffolds manufactured by an engineering approach to fabricate a potential tissue engineering product that provides both new tissue formation and wound healing. Thus, Hypericum perforatum oil (HPO) and vitamin A palmitate (VAP) incorporated gelatin (Gt) nanofibrous scaffolds were successfully prepared by the electrospinning method. Tannic acid (TA) was used as the cross-linking agent. The amounts of VAP and HPO loaded in the base Gt solution [15% w/v in 4:6 v/v acetic acid/deionized water] were 5 and 50 wt % (based on the weight of Gt), respectively. The obtained scaffolds were studied regarding their microstructure, chemical structure, thermal stability, antibacterial activity, in vitro release study, and cellular proliferation assay. In the light of these studies, it was determined that VAP and HPO were incorporated successfully in Gt nanofibers cross-linked with TA. Release kinetic tests confirmed that the patterns of TA and VAP release were consistent with the Higuchi model, whereas HPO release was consistent with the first-order kinetic model. In addition, this membrane was biocompatible with L929 fibroblast cells and had antibacterial activity and thermal stability. This preliminary study suggests potential applicability of the proposed dressing to treat skin wounds in clinics.

Three-dimensional culture conditioned bone marrow MSC secretome accelerates wound healing in a burn injury mouse model

Mesenchymal stem cell (MSC)-based therapy has emerged as a promising regenerative therapeutic approach for wound healing. To determine the effects of cultured MSCs as a 2D monolayer (2D-MSCs) and 3D spheroids (3D-MSCs) on their secretomes, and to examine the effect of 3D-MSC secretomes on endothelial cells (ECs) and MSCs in a burn injury mouse model. MSCs were cultured as 2D monolayers (2D-MSCs) and 3D spheroids (3D-MSCs) and their cellular characteristics were evaluated by western blotting. 2D-MSC and 3D-MSC secretomes (condition medium: CM) were analyzed using an angiogenic array. The activation of ECs by 2D-MSC and 3D-MSC CMs was examined in cellular proliferation, migration, and tube formation assays. The wound healing effects of 2D-MSCs and 3D-MSCs were determined in vivo using a burn injury mouse model. 3D culture conditions altered the markers of components that regulate cell survival, cytoskeletal, adhesion, and proliferation. Interleukin-6 (IL-6), vascular endothelial growth factor A (VEGFA), IL-8, and chemokine (CXC motif) ligand 1 (CXCL1) were present at high levels in the CM of 3D-MSCs compared with 2D-MCs. 3D-MSC-CMs promoted the proliferation, migration, and tube formation of ECs. Furthermore, 3D-MSC treatment enhanced wound healing in a burn injury mouse model. 3D culture improves proangiogenic factors in the MSC secretome and 3D-MSCs represent a new cell-based treatment strategy for wound healing.

Regorafenib inhibits growth, survival and angiogenesis in nasopharyngeal carcinoma and is synergistic with Mcl-1 inhibitor.

Regorafenib is an oral multi-kinase inhibitor approved for various metastatic/advanced cancers, and has been investigated in clinical trials in many other tumour entities. The purpose of this study was to evaluate the therapeutic potential of regorafenib for nasopharyngeal carcinoma (NPC). Cellular proliferation, survival, apoptosis and colony formation assays were performed and combination index was determined. NPC xenograft tumour models were established. In vitro and In vivo angiogenesis assays were performed. Regorafenib is effective against a panel of NPC cell lines regardless of cellular origin and genetic profiling while sparing normal nasal epithelial cells. The predominant inhibitory effects of regorafenib in NPC are anchorage-dependent and anchorage-independent growth rather than survival. Apart from tumour cells, regorafenib potently inhibits angiogenesis. Mechanistically, regorafenib inhibits multiple oncogenic pathways including Raf/Erk/Mek and PI3K/Akt/mTOR. Regorafenib decreases Bcl-2 but not Mcl-1 level in NPC cells. The in vitro observations are evident in in vivo NPC xenograft mouse model. The combination of Mcl-1 inhibitor with regorafenib is synergistic in inhibiting NPC growth without causing systemic toxicity in mice. Our findings also support further clinical investigation of regorafenib and Mcl-1 inhibitor for NPC treatment.

Abstract 503: ISM3412, a novel and selective MAT2A inhibitor for the treatment of cancer

Abstract The enzyme methionine adenosyltransferase 2α (MAT2A) has an important role in metabolism and epigenetics as the major producer of the universal methyl donor S-adenosyl methionine (SAM). Despite broad cellular functions, MAT2A was recently implicated as a synthetic lethal target in cancers that have a deletion of the methylthioadenosine phosphorylase (MTAP) gene, which is found in approximately 15% of all cancers. Here, we report ISM3412 is a novel potent MAT2A inhibitor with high selectivity. In enzymatic assays, ISM3412 demonstrated a strong inhibition of MAT2A with an IC50 of 19.4 nM. In both HCT116 MTAP-/- cells and HCT116 parental cells, the compound inhibited SAM production, with IC50 values of 5.97 nM and 3.66 nM, respectively. Additionally, it inhibited SDMA production in HCT116 MTAP-/- cells (IC50 of 6.37 nM), but not in HCT116 parental cells (IC50 &amp;gt; 2000 nM). Consistent with the large SDMA inhibitory potency difference between the HCT116 isogenic cell pair, ISM3412 also exhibited very good selectivity in cellular proliferation assays. While it inhibited cellular growth in HCT116 MTAP-/-cells with an IC50 of 199nM, its inhibitory potency on HCT116 parental cells was very weak (IC50 of &amp;gt;21117nM), revealing more than a 100-fold selectivity in inhibiting growth in this isogenic pair cell line. Furthermore, while ISM3412 strongly inhibited proliferation in multiple cancer cell lines with natural MTAP deficiency (including pancreatic cancer, bladder cancer, and NSCLC), the same was not observed in multiple MTAP-intact cell lines (IC50 &amp;gt; 30 uM). Consistent with the in vitro data, ISM3412 demonstrated robust mono-therapeutic efficacy in an MTAP-/- HCT116 CDX model with tumor growth inhibition of 66% at 3 mg/kg QD. Similarly, ISM3412 also demonstrated robust single-agent efficacy in CDX models of pancreatic cancer, bladder cancer and NSCLC, as well as in a PDX model of esophageal cancer. Notably, ISM3412 showed improved efficacy when combined with docetaxel, compared to the individual monotherapy. ISM3412, at 10 uM, showed no obvious agonistic or antagonistic effect on 44 selected targets in a mini safety panel, demonstrating its selectivity as a MAT2A inhibitor. In addition to its strong biological potency and selectivity, ISM3412 exhibited favorable drug-likeness properties, including low molecular weight, good solubility and permeability, and low plasma protein binding. In vivo PK data revealed low clearance and high oral-bioavailability. Further, it was well tolerated with no significant hepatobiliary toxicity. In conclusion, ISM3412 is a novel and highly selective MAT2A inhibitor for MTAP-deficient cancers, and merits clinical evaluation. Citation Format: Qingyuan Meng, Xiao Ding, Xiaosong Liu, Hailong Wang, Ling Wang, Junwen Qiao, Hua Cao, Yushu Yin, Congcong Zhu, Xing Liang, Xin Cai, Yuanyuan Xu, Chenxi Xu, Shan Chen, Sujata Rao, Feng Ren, Alex Zhavoronkov. ISM3412, a novel and selective MAT2A inhibitor for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 503.

LMP1 mediates tumorigenesis through persistent epigenetic modifications and PGC1β upregulation.

Latent membrane protein 1 (LMP1), which is encoded by the Epstein‑Barr virus (EBV), has been considered as an oncogene, although the detailed mechanism behind its function remains unclear. It has been previously reported that LMP1 promotes tumorigenesis by upregulation of peroxisome proliferator‑activated receptor‑γ coactivator‑1β (PGC1β). The present study aimed to investigate the potential mechanism for transient EBV/LMP1 exposure‑mediated persistent PGC1β expression and subsequent tumorigenesis through modification of mitochondrial function. Luciferase reporter assay, chromatin immunoprecipitation and DNA mutation techniques were used to evaluate the PGC1β‑mediated expression of dynamin‑related protein 1 (DRP1). Tumorigenesis was evaluated by gene expression, oxidative stress, mitochondrial function and invitro cellular proliferation assays. The potential effects of EBV, LMP1 and PGC1β on tumor growth were evaluated in an invivo xenograft mouse model. The present invitro experiments showed that LMP1 knockdown did not affect PGC1β expression or subsequent cell proliferation in EBV‑positive tumor cells. PGC1β regulated DRP1 expression by coactivation of GA‑binding protein α and nuclear respiratory factor 1 located on the DRP1 promoter, subsequently modulating mitochondrial fission. Transient exposure of either EBV or LMP1 in human hematopoietic stem cells caused persistent epigenetic changes and PGC1β upregulation after long‑term cell culture even in the absence of EBV/LMP1, which decreased oxidative stress, and potentiated mitochondrial function and cell proliferation invitro. Enhanced tumor growth and shortened survival were subsequently observed invivo. It was concluded that PGC1β expression and subsequent cell proliferation were independent from LMP1 in EBV‑positive tumor cells. PGC1β modulated mitochondria fission by regulation of DRP1 expression. Transient EBV/LMP1 exposure caused persistent PGC1β expression, triggering tumor growth in the absence of LMP1. The present study proposes a novel mechanism for transient EBV/LMP1 exposure‑mediated tumorigenesis through persistent epigenetic changes and PGC1β upregulation, uncovering the reason why numerous forms of lymphoma exhibit upregulated PGC1β expression, but are devoid of EBV/LMP1.

Review on Nanoparticle Toxicity and their Methods of Assessment in Humans

Nanoparticles, also known as zero-dimensional particles, are tiny, solid, colloidal forms of matter with diameters ranging from 1nm to 102 nm. Engineering nanoparticles (NPs) at the atomic scale (100 nm in diameter) has produced a number of unique and practical uses in a variety of fields, including electronics, chemistry, environmental protection, medical imaging, illness diagnosis, drug delivery, and cancer. This review aims to analyse potential toxicological portal routes connected to NPs exposures in order to better understand the effects of these exposures on health and how to create appropriate monitoring and control techniques. In actuality, the skin pores, weakened tissues, injection, olfactory, respiratory, and digestive tracts are all entry points for these ultrafine particles into the body. One of the mechanisms of NPs' toxicity is ROS production, which can result in oxidative stress, inflammation, and subsequent damage to proteins, cell membranes, and DNA. This review includes information on several types of nanoparticle toxicity, including neurotoxicity, genotoxicity, ocular toxicity, and dermal toxicity. This study aims to outline techniques for evaluating the toxicity of nanoparticles, including in-vitro techniques such size and surface charge evaluation, cellular interaction test, proliferation assay, apoptosis assay, necrosis assay, and DNA Assay, Endotoxin, Oxidative Stress, and Damage Assay and in vivo techniques including Hematology, serum chemistry, histopathology, and biodistribution and clearance. The review also discusses measures that can be implemented to reduce the toxicity of nanoparticles

ERRATUM: Biological activities of green coffee nanoemulsions evaluated through alternative methods: MTT, cellular proliferation, and HET-CAM assays

ERRATUM: Biological activities of green coffee nanoemulsions evaluated through alternative methods: MTT, cellular proliferation, and HET-CAM assays

Biological activities of green coffee nanoemulsions evaluated through alternative methods: MTT, cellular proliferation, and HET-CAM assays

Biological activities of green coffee nanoemulsions evaluated through alternative methods: MTT, cellular proliferation, and HET-CAM assays

Potential role of Acanthamoeba Rab7

Acanthamoeba castellanii is a free-living protozoan that causes several severe human parasitic diseases such as Acanthamoeba keratitis and granulomatous encephalitis. A. castellanii feeds on bacteria, yeasts, and other organic particles as food sources, but the mechanisms of digestion in acanthamoebal cells are unclear. Rab GTPases participate in endosomal delivery in eukaryotes after phagocytosis. This study aimed to determine the potential functions of A. castellanii Rab7 (AcRab7), which is involved in phagocytosis, and the relationship between AcRab7 and further cellular physiological phenomena. In this study, the inhibitor CID1067700 (CID) was used to specifically inhibit the binding of nucleotides to confirm the potential functions of AcRab7. Cellular proliferation and ATP assays were also used to detect underlying cellular physiological functions after blocking the phagocytosis pathway. We found that AcRab7 expression increased as the co-culture time with Escherichia coli increased. Immunofluorescence staining showed that AcRab7 colocalized with lysosomes in its GTP-activating form. In addition, AcRab7 inhibition resulted in a reduction in cell proliferation and ATP levels. Our results suggest that AcRab7 participates in endosomal delivery and dominates energy production and cell growth.

Abstract 745: Molecular landscape of arsenic-induced bladder carcinogenesis

Abstract Introduction: Epidemiological, pre-clinical, and clinical studies have linked chronic arsenic (sodium arsenite: NaAsO2) exposure to a myriad of adverse health dysfunctions including the development of bladder cancer (BCa). However, most of the studies on arsenic-induced BCa have been performed on either unsuitable cell lines or using a higher concentration of arsenic (mM), which does not reflect exposure levels in a real-world setting. We used the telomerase reverse transcriptase (HU-hTERT1) immortalized normal bladder epithelial and a median physiological concentration of NaAsO2 (250 nM: 33 mg/L), to determine the transcriptomic and phenotypic changes during malignant transformation of HU-hTERT1 cells. Methods: HU-hTERT1 cells were exposed to 250nM arsenic for 12 months. To analyze the effect of arsenic exposure we performed cell viability, cellular proliferation, and clonogenic assays. RNA Seq data was used to perform pathway enrichment analysis, Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and protein-protein interaction (PPI) studies. Results: BCa patients had significantly higher (4-5 fold) levels of arsenic (20-50 mg/L) in their urine when compared to their healthy counterparts (&amp;lt;8 mg/L). Acute exposure to high concentration of (&amp;gt; 400nM) inhibited the HU-hTERT1 growth, whereas concentrations less than 400nM did not adversely affect the viability of these cells. HU-hTER1 cells began forming colonies after six months of chronic NaAsO2 exposure, with the number of colonies increasing concurrently with the exposure time. GO and KEGG analysis showed a total of 2174 genes that are differentially expressed in arsenic-treated HU-hTER1 transforming cells (0 vs 12 months). The upregulated genes (1392) were involved in cellular response to unfolded protein, response to endoplasmic reticulum stress, proteasome-mediated ubiquitin-dependent protein degradation, autophagy signaling, and tumor angiogenesis(P&amp;lt;0.05). Interestingly, the PPI analysis demonstrated a significant upregulation in histone modification of genes. These genes were enriched for the binding sites of transcription factors that are key regulators of carcinogenic pathways including ataxia-telangiectasia mutated (ATM) kinase and cell cycle regulatory pathways demonstrating a novel link between NaAsO2 exposure and BCa. Conclusion: An in vitro model of NaAsO2-induced malignant transformation was developed. Identifying precise molecular events that lead to arsenic-induced malignant transformation of normal urothelial cells would be an important step in defining therapy for BCa. Citation Format: Ajit Kumar Navin, Vaibhav Shukla, Balaji Chandrasekaran, Ashish Tyagi, Uttara Saran, Murali K. Ankem, Chendil Damodaran. Molecular landscape of arsenic-induced bladder carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 745.

Galunisertib Exerts Antifibrotic Effects on TGF-β-Induced Fibroproliferative Dermal Fibroblasts.

Dermal fibroblasts in pathological scars secrete constitutively elevated levels of TGF-β, signaling the transcription of fibrotic genes via activin-like kinase 5 (ALK5). In the present study, we examine the antifibrotic effects of galunisertib, a small-molecule inhibitor of ALK5, on fibroproliferative dermal fibroblasts in an in vitro model of wound healing. We induced fibrosis in human dermal fibroblasts with exogenous TGF-β and performed cellular proliferation assays after treatment with varying concentrations of galunisertib. Dermal fibroblast proliferation was diminished to homeostatic levels without cytotoxicity at concentrations as high as 10 μM. An in vitro scratch assay revealed that galunisertib significantly enhanced cellular migration and in vitro wound closure beginning 24 h post-injury. A gene expression analysis demonstrated a significant attenuation of fibrotic gene expression, including collagen-1a, alpha-smooth muscle actin, fibronectin, and connective tissue growth factor, with increased expression of the antifibrotic genes MMP1 and decorin. Protein synthesis assays confirmed drug activity and corroborated the transcription findings. In summary, galunisertib simultaneously exerts antifibrotic effects on dermal fibroblasts while enhancing rates of in vitro wound closure. Galunisertib has already completed phase II clinical trials for cancer therapy with minimal adverse effects and is a promising candidate for the treatment and prevention of pathological cutaneous scars.

Abstract 216: Expression of DNA damage response proteins modifies the efficacy of CDK4/6 inhibitor-mediated radiosensitization in breast cancer models

Abstract Purpose: CDK4/6 inhibitors (CDK4/6i) are standard of care for the treatment of locally advanced and metastatic estrogen receptor-positive (ER+), HER2-negative metastatic breast cancer (BC). CDK4/6 inhibition + radiation therapy (RT) is synergistic in both ER+ and triple negative breast cancers (TNBC), but the underlying mechanism is not entirely understood. In this study, we evaluated how pre-existing or genetically engineered deficits in DNA damage response genes (BRCA1/2, RAD51, RB1, XRCC6, TP53) influence radiosensitization. We hypothesized that inhibition of homologous recombination (HR) would prevent CDK4/6i-mediated radiosensitization and blocking non-homologous end joining (NHEJ) would be synergistic. Methods: Cellular proliferation assays determined the half-maximal inhibitory concentrations (IC50) of the 3 approved CDK4/6i palbociclib, ribociclib, and abemaciclib. Clonogenic survival assays determined the radiation enhancement ratios (rERs) and evaluated the efficacy of CDK4/6i + RT. Immunofluorescence assays measured RAD51 foci formation and quantified micronuclei formation following RT and/or CDK4/6 inhibition. Immunoprecipitation with myc-RAD51 and GFP-RB assessed potential protein-protein interactions. Results: While ER+ and TNBC cell lines with wild type BRCA1 expression are radiosensitized by CDK4/6i, BRCA1-deficient SUM-149 cells are not radiosensitized by CDK4/6i at concentrations up to 1µM (rER: 0.92-1.01). In an MCF-7 isogenic model of BRCA2 knockout, CDK4/6i-mediated radiosensitization was abolished compared to Cas9 control or parental cell lines. In ER+ BC cell lines (MCF-7-p53 wt, T47D-p53 mutant), transient or genetic knockdown of RAD51 prevented CDK4/6i-induced radiosensitization. The total quantity of RT-induced RAD51 foci increased in vitro following overexpression of RB-a tumor suppressor and downstream target of CDK4/6. RB overexpression also rescued CDK4/6i-mediated radiosensitization in RB-deficient cell lines through changes in HR efficiency but not via NHEJ or altered micronuclei formation. Moreover, immunoprecipitation of RAD51 in ER+ (MCF-7) and TNBC (MDA-MB-231) cells exhibited an interaction with RB. Conversely, loss of the NHEJ-associated protein Ku70 (XRCC6) was synergistic with palbociclib + RT in MCF7 (rER: 1.76-2.44) and T47D (rER: 1.61-3.88) cells. Finally, CRISPR Cas9-mediated loss of the tumor suppressor p53 (TP53) did not affect radiosensitization induced by CDK4/6i in isogenic p53 wt ER+ (MCF-7, rER: 1.19-1.33) and p53 wt TNBC (CAL-51, rER: 1.23-1.52) cell lines with TP53 loss. Conclusions: Taken together, our results in multiple non-overlapping isogenic models of ER+ and TNBC suggest that CDK4/6i-mediated radiosensitization of BC cell lines occurs through impaired HR activity and RB signaling, and not through the actions of p53 or NHEJ-mediated DNA repair. Citation Format: Kassidy M. Jungles, Andrea M. Pesch, Nicole Hirsh, Anna R. Michmerhuizen, Kari Wilder-Romans, Benjamin C. Chandler, Meilan Liu, Lynn Lerner, Lori J. Pierce, James M. Rae, Corey W. Speers. Expression of DNA damage response proteins modifies the efficacy of CDK4/6 inhibitor-mediated radiosensitization in breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 216.