Clonogenic Assay Research Articles

Abstract A011: Enhanced Radiosensitivity of Pancreatic Cancer Achieved through Inhibition of Cyclin-Dependent Kinase 1

Abstract Introduction: One of the major challenges in treating Pancreatic Ductal Adenocarcinoma (PDAC) is overcoming radioresistance. This study investigates the targeting of Cyclin-dependent kinase-1 (CDK1) through both genetic and pharmaceutical inhibition as a strategy to increase the radiosensitivity of PDAC cells. Methods: We conducted mass spectrometry and phosphoproteomics analyses to compare engineered radiation-resistant PDAC cell lines (MIA PaCa-2 and PANC-1) with their parental controls. Additionally, we examined the TCGA PDAC database to correlate clinical outcomes, such as overall survival (OS), with CDK1 mRNA expression levels in PDAC patients. Furthermore, we developed a microRNA-based TET-on inducible shRNA to specifically inhibit CDK1 expression in PDAC cell lines. In an orthotopic PDAC model, we evaluated the radiation sensitivity of PDAC tumors with or without doxycycline treatment. We also employed a selective CDK1 inhibitor, RO-3306, to target CDK1 activation, followed by in vitro experiments using immunoblotting, immunocytochemistry, and clonogenic assays. Results: Our phosphoproteomics analysis revealed a significant increase in phospho-CDK1 (Tyr15) levels in the radiation-resistant cell lines. High CDK1 expression was associated with poorer OS in PDAC patients. Additionally, we observed heightened CDK1 phosphorylation at the threonine tyrosine 14 (Tyr14) residue following radiation exposure. Through our in vivo and in vitro experiments, we demonstrated that CDK1 inhibition, in conjunction with radiation therapy, delayed tumor growth and enhanced radiosensitivity in PDAC cells. Treatment with RO-3306 caused a substantial shift of cells into the G2/M phase and disrupted DNA repair post-radiation exposure, further enhancing radiosensitivity. Conclusion: Our findings underscore the critical role of CDK1 in PDAC radioresistance. Targeting CDK1 in combination with radiotherapy shows promise for future exploration as a potential treatment strategy in PDAC. Citation Format: Stetson Van Matre, Saaimatul Huq, Lokesh Akana, Oscar Zuniga, Henrique Rodrigues, Adam Wolfe. Enhanced Radiosensitivity of Pancreatic Cancer Achieved through Inhibition of Cyclin-Dependent Kinase 1 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A011.

Phytochemical Profile of Cymbopogon citratus (DC.) Stapf Lemongrass Essential Oil from Northeastern Thailand and Its Antioxidant and Antimicrobial Attributes and Cytotoxic Effects on HT-29 Human Colorectal Adenocarcinoma Cells.

Colorectal cancer is the third most prevalent cancer in Thailand, prompting the search for alternative or preventive treatments using natural constituents. In this study, the authors employed hydrodistillation to extract Cymbopogon citratus (DC.) Stapf (lemongrass) essential oil (LEO) from plants in northeastern Thailand and assessed its chemical profile, antioxidant, antimicrobial, and anticancer properties. The LEO displayed potent antioxidant activities in DPPH and hydroxyl scavenging assays with IC50 values of 2.58 ± 0.08 and 4.05 ± 0.12 mg/mL, respectively, and demonstrated antimicrobial activities against Escherichia coli, Cutibacterium acnes, Streptococcus agalactiae, and Staphylococcus aureus at 8-10 µg/mL. At 48 h, the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay showed the LEO exhibiting low cell viability (3%) at concentrations of 200-400 µg/mL, with an IC50 value of 82.46 ± 1.73 µg/mL, while in the clonogenic assay it exhibited a lower IC50 value of 23.11 ± 1.80 µg/mL. The GC-MS analysis identified citral (79.24%) consisting of 44.52% geranial and 34.72% neral, and β-myrcene (5.56%). The addition of LEO significantly influenced apoptotic genes (Bcl-2, Bax, p21, and Caspase-3) and proteins, as indicated by real-time polymerase chain reaction (RT-PCR) and Western blot studies. Results suggested that LEO initiated apoptosis through intrinsic pathways and demonstrated potential as a chemopreventive, antimicrobial, and antioxidant agent with substantial health advantages.

Senegenin suppresses hepatocellular carcinoma by regulating O-GlcNAcylation.

Based on current knowledge, hepatocellular carcinoma (HCC) is a condition with numerous etiologies and risk factors. However, the pathogenesis of HCC remains unclear. To investigate the roles of senegenin and O-GlcNAcylation in the growth and metastasis of HCC. The levels of O-linked N-acetylglucosamine transferase (OGT) and O-GlcNAcylation in HCC cells and tissues were detected using western blot analysis. The effects of senegenin and O-GlcNAcylation on the proliferation of HCC cells were investigated in vitro using cell counting kit-8 and clonogenic assays. The potential effects of senegenin and O-GlcNAcylation on HCC metastasis were examined using the transwell migration assay. O-GlcNAcylation levels were altered via drug treatment and lentiviral infection, and western blot analysis was used to detect proteins involved in various pathways. Western blot analysis revealed that OGT and O-GlcNAcylation levels were significantly elevated in HCC tissues and cells. O-GlcNAcylation levels in HCC cells were significantly altered by drug treatment and lentiviral infection. An increase in the glycosylation level was linked to enhanced proliferation, invasiveness, clonogenicity, and metastatic potential of cancer cells. O-GlcNAcylation induced by senegenin was found to slow the proliferation and migration of HCC cells. The levels of proteins involved in nuclear factor-kappa B (NF-κB) and c-Jun N-terminal kinase (JNK) pathways, which are associated with endoplasmic reticulum stress, were altered. Senegenin lowers O-GlcNAcylation levels, decreases OGT expression, and inhibits cancer cell growth and metastasis by regulating proteins involved in NF-κB and JNK pathways.

Abstract B089: Identification of novel drivers of gemcitabine resistance in pancreatic cancer

Abstract Pancreatic cancer is an aggressive disease with limited therapeutic options. Gemcitabine is a clinically used chemotherapy for pancreatic cancer treatment but has limited efficacy. The molecular mechanisms responsible for gemcitabine resistance in pancreatic cancer are still unclear. Here, we analyzed the genomics of drug sensitivity in cancer dataset and identified various pancreatic cancer cells with distinct sensitivity to gemcitabine. Validation with proliferation and clonogenic assays confirmed that while CFPAC1 and PANC0327 are highly gemcitabine-sensitive, ASPC1 and PANC1 display intrinsic gemcitabine resistance. Analysis of the gene expression profile of these cells identified several genes that are differentially upregulated in resistant versus sensitive cells. Pathway enrichment analysis using the differential genes revealed the upregulation of Hippo, Wnt, MAPK signaling pathways as well as autophagy and glutathione metabolism in the resistant cells. Using quantitative PCR, immunoblotting and biochemical assays, and extracellular flux analysis methods, we have characterized the molecular distinctions between gemcitabine sensitive and resistant cells. Our results provide insight on the molecular mechanisms driving gemcitabine resistance in pancreatic cancer and could lead to novel therapeutic strategies to overcome resistance and improve patients’ treatment. Citation Format: Amina D Bilalbegovic, Bowen Fu, Zeribe Nwosu. Identification of novel drivers of gemcitabine resistance in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B089.

Enhanced radiosensitivity of pancreatic cancer achieved through inhibition of Cyclin-dependent kinase 1

Background and purposeOvercoming radioresistance is a critical challenge in pancreatic ductal adenocarcinoma (PDAC). Our study investigates the targeting of Cyclin-dependent kinase-1 (CDK1) through genetic and pharmaceutical inhibition to radiosensitize PDAC cells. Materials and MethodsMass spectrometry and phosphoproteomics were used to analyze engineered radiation-resistant PDAC cell lines (MIA PaCa-2 and PANC-1) compared to parental controls. The TCGA PDAC database was queried for clinical outcomes and patients were dichotomized based on the median CDK1 mRNA expression. We generated a microRNA-based TET-on inducible shRNA to inhibit CDK1 expression in two PDAC cell lines. We used an orthotopic model of PDAC to test the radiation sensitivity of PDAC tumors with or without doxycycline treatment. We targeted CDK1 activation with a selective CDK1 inhibitor, RO-3306, followed by in vitro experiments employing immunoblotting, immunocytochemistry, and clonogenic assays. ResultsPhosphoproteomics analysis revealed that phospho-CDK1 (Tyr15) was significantly elevated in the resistant clones. We found that high CDK1 expression was associated with worse OS in PDAC patients. Radiation exposure increased CDK1 phosphorylation. In MIA PaCa-2 and PANC-1 cells, CDK1 inhibition synergized with radiation therapy to delay tumor growth in vivo. CDK1 inhibition via. RO-3306 resulted in a significant shift of cells into the G2/M phase and disrupted DNA repair after radiation exposure. In vitro, pre-treatment with RO-3306 led to enhanced radiosensitivity of PDAC cells. ConclusionCDK1 plays a crucial role in PDAC radioresistance. Targeting CDK1 with radiotherapy holds promise for further investigation in PDAC treatment.

Connexin 25 maintains self-renewal and functions of airway basal cells for airway regeneration

BackgroundThe formation of stem cell clones enables close contact of stem cells inside. The gap junctions in such clone spheres establish a microenvironment that allows frequent intercellular communication to maintain self-renewal and functions of stem cells. Nevertheless, the essential gap junction protein for molecular signaling in clones is poorly known.MethodsPrimary human airway basal cells (hBCs) were isolated from brushing samples through bronchoscopy and then cultured. A tightly focused femtosecond laser was used to excite the local Ca2+ in an individual cell to initiate an internal Ca2+ wave in a clone to screen gap junction proteins. Immunoflourescence staining and clonogenicity assay were used to evaluate self-renewal and functions. RNA and protein levels were assessed by PCR and Western blot. Air–liquid interface assay was conducted to evaluate the differentiation potential. A Naphthalene injury mouse model was used to assess the regeneration potential.ResultsHerein, we identify Connexin 25 (Cx25) dominates intercellular Ca2+ communications in clones of hBCs in vitro to maintain the self-renewal and pluripotency of them. The self-renewal and in vitro differentiation functions and in vivo regeneration potential of hBCs in an airway damage model are both regulated by Cx25. The abnormal expression of Cx25 is validated in several diseases including IPF, Covid-19 and bronchiectasis.ConclusionCx25 is essential for hBC clones in maintaining self-renewal and functions of hBCs via gap junctions.

A comparative study of sensitizers and liposome composition in radiation-induced controlled drug release for cancer therapy

This study investigates drug-loaded liposomes designed for controlled release under ionizing radiation to refine cancer treatment precision. Liposomes as carriers enable targeted chemotherapy delivery, reducing healthy tissue damage risk. Liposomes containing poly- or mono-unsaturated fatty acids and various sensitizing agents were assessed for responsiveness to UV light and γ photon irradiation including rose bengal (RB), protoporphyrin IX (PPIX), verteporfin (VP), cercosporin (CERC) and hypericin (HYP). Carboxyfluorescein (CF) was used as a surrogate for drug release measurements. VP and PPIX induced rapid drug release and lipid peroxidation under UV light, while RB prompted quick drug release under UV light and a modest immediate release under γ irradiation, eventually reaching full release a few hours after irradiation, demonstrating dose-dependent effects. Smaller liposomes displayed accelerated release, emphasizing size-dependent kinetics. In vitro analyses evaluated radiosensitizing effects of RB-loaded liposomes. Clonogenic assays indicated that RB-filled liposomes had minimal direct radiobiological effects but increased indirect radiation damage, as shown by the curvature of the cell survival curve. Our study sheds light on factors influencing liposomal drug release under ionizing radiation, spotlighting RB as a promising radiosensitizer requiring further investigation for cancer therapy potential.

Evaluation of the effects of vitamin D analogs, bevacizumab, and radiotherapy in uveal melanoma cells

Due to the lack of a definitive effective treatment method that provides a complete cure and increases survival rates in uveal melanoma, the search for alternative treatments at the molecular level continues. In this context, we aimed to comparatively analyze the therapeutic effects of 25-hydroxyvitamin D3 (D2), 1a, 25-dihydroxyvitamin D3 (D3), bevacizumab and radiotherapy (RT) in a uveal melanoma cell line (MP41). Cytotoxicity was evaluated using XTT cell proliferation kit and Xcelligence cell analyzer system. RT dose was determined after a clonogenic assay. Annexin V/PI staining and Western blot analyses for caspase-3, -8, and -9 were performed to analyze apoptosis. Additionally, cell cycle analyses were also conducted. As a result, we found that D2 and D3 did not show cytotoxic effects, while bevacizumab and RT showed time and dose-dependent cytotoxicity. IC50 concentration of bevacizumab was 6.945 mg/mL. Radiotherapy and bevacizumab significantly reduced cell survival and induced apoptosis when administered both as monotherapy and in combination. A significant increase in caspase proteins was detected at high bevacizumab concentrations. However, the combination of bevacizumab and radiotherapy caused a substantial decrease in caspase-3, -8 and -9 expressions. No significant difference in cell cycle distribution was detected in any treatment. Our results showed that bevacizumab inhibited MP41 cell proliferation and had an additive effect when administered with RT. In conclusion, our study offers a different perspective on the treatment of uveal melanoma, and these results, when supported by animal experiments and clinical studies in the future, might be a new step in the treatment of this challenging ocular tumor.

PRMT1 promotes radiotherapy resistance in glioma stem cells by inhibiting ferroptosis.

The existence of glioma stem cells (GSCs) in cancer is related to glioma radiotherapy resistance. In this research, the effect of protein arginine methyltransferase 1 (PRMT1) on the radiosensitivity of glioma stem cell (GSC)-like cells, as well as its underlying mechanism, was investigated. GSCs-like cells were analyzed and identified by flow cytometry. The self-renewal capability was evaluated by sphere-forming assay. The PRMT1 expression level in glioblastoma were analyzed using the Gene Expression Profiling Interactive Analysis database. The mRNA and protein were scrutinized by RT-qPCR and western blot, respectively. The radiosensitivity was evaluated by clonogenic survival assay. Ferroptosis was evaluated by detecting the levels of reactive oxygen species, malondialdehyde, Fe2+, glutathione, and 4-hydroxynonenal. U87 and SHG44 cells with GSC-like phenotype (GSC-U87 and GSC-SHG44) displayed strong expression of CD133 and nestin versus the glioma cells. GSC-U87 and GSC-SHG44 possess the self-renewal capability. The level of PRMT1 was higher in glioblastoma tumor tissues than in the normal paracancer tissues. Knockdown of PRMT1 enhanced the radiotherapy sensitivity of GSCs-like cells, which wasevidenced by reduced survival fraction in GSC-U87 and GSC-SHG44 underwent sh-PRMT1 transfection. But, this effect was attenuated by Fer-1 (a ferroptosis inhibitor) treatment, accompanied by the abatement of ferroptosis. PRMT1 promoted radiotherapy resistance in GSCs-like cells by inhibiting ferroptosis.

Isolation and comparative genotoxicity screening of trichokonins VI and VIII on CHO-K1 cells

Peptaibols are fungal peptides that exhibit efficacy against pathogen microorganisms. Trichokonin VI (TK-VI) and trichokonin VIII (TK-VIII) are known peptaibols isolated from the endolichenic fungi Hypocrea sp. Previous investigations reported that trichokonin VI presents antiproliferative effects on tumor cells. This study is pioneering in elucidating the genotoxic effects of TK-VI and TK-VIII, contributing to the thorough assessment of their safety as potential therapeutic agents. The present investigation aimed to evaluate the genotoxicity of TK-VI and TK-VIII on CHO-K1 cells. Cytotoxicity was evaluated using the XTT assay and clonogenic survival assays, followed by evaluation of DNA damage using the comet assay and micronucleus test conducted in vitro. The XTT assay results indicated IC50 values of 10.30 µM and 9.89 µM for TK-VI and TK-VIII, respectively. The clonogenic survival assay indicated that concentrations of 10 µM or higher completely inhibited the cell colony formation. In the comet assay, both TK-VI and TK-VIII increased the DNA damage score and the frequency of comet nuclei in all tested concentrations. In the micronucleus assay, TK-VI and TK-VIII at 10 µM increased the frequency of MN in CHO-K1 cells. Both TK-VI and TK-VIII exhibited genotoxic effects. Our findings underscore the importance of considering the genotoxicological safety of peptaibols, particularly when assessing their potential for other biological activities.

FACT inhibitor CBL0137, administered in an optimized schedule, potentiates radiation therapy for glioblastoma by suppressing DNA damage repair.

Standard-of-care for glioblastoma remains surgical debulking followed by temozolomide and radiation. However, many tumors become radio-resistant while radiation damages surrounding brain tissue. Novel therapies are needed to increase the effectiveness of radiation and reduce the required radiation dose. Drug candidate CBL0137 is efficacious against glioblastoma by inhibiting histone chaperone FACT, known to be involved in DNA damage repair. We investigated the combination of CBL0137 and radiation on glioblastoma. In vitro, we combined CBL0137 with radiation on U87MG and A1207 glioblastoma cells using the clonogenic assay to evaluate the response to several treatment regimens, and the Fast Halo Assay to examine DNA repair. In vivo, we used the optimum combination treatment regimen to evaluate the response of orthotopic tumors in nude mice. In vitro, the combination of CBL0137 and radiation is superior to either alone and administering CBL0137 two hours prior to radiation, having the drug present during and for a prolonged period post-radiation, is an optimal schedule. CBL0137 inhibits DNA damage repair following radiation and affects the subcellular distribution of histone chaperone ATRX, a molecule involved in DNA repair. In vivo, one dose of CBL0137 is efficacious and the combination of CBL0137 with radiation increases median survival over either monotherapy. CBL0137 is most effective with radiation for glioblastoma when present at the time of radiation, immediately after and for a prolonged period post-radiation, by inhibiting DNA repair caused by radiation. The combination leads to increased survival making it attractive as a dual therapy.

FACT inhibitor CBL0137, administered in an optimized schedule, potentiates radiation therapy for glioblastoma by suppressing DNA damage repair.

Standard-of-care for glioblastoma remains surgical debulking followed by temozolomide and radiation. However, many tumors become radio-resistant while radiation damages surrounding brain tissue. Novel therapies are needed to increase the effectiveness of radiation and reduce the required radiation dose. Drug candidate CBL0137 is efficacious against glioblastoma by inhibiting histone chaperone FACT, known to be involved in DNA damage repair. We investigated the combination of CBL0137 and radiation on glioblastoma. In vitro, we combined CBL0137 with radiation on U87MG and A1207 glioblastoma cells using the clonogenic assay to evaluate the response to several treatment regimens, and the Fast Halo Assay to examine DNA repair. In vivo, we used the optimum combination treatment regimen to evaluate the response of orthotopic tumors in nude mice. In vitro, the combination of CBL0137 and radiation is superior to either alone and administering CBL0137 two hours prior to radiation, having the drug present during and for a prolonged period post-radiation, is an optimal schedule. CBL0137 inhibits DNA damage repair following radiation and affects the subcellular distribution of histone chaperone ATRX, a molecule involved in DNA repair. In vivo, one dose of CBL0137 is efficacious and the combination of CBL0137 with radiation increases median survival over either monotherapy. CBL0137 is most effective with radiation for glioblastoma when present at the time of radiation, immediately after and for a prolonged period post-radiation, by inhibiting DNA repair caused by radiation. The combination leads to increased survival making it attractive as a dual therapy.

Simvastatin-loaded gelatin-capped polycaprolactone nanoparticles for the post-operative management of breast cancer

Minimal residual disease at the tumor site after surgical ablation is a major cause for tumor recurrence and metastasis. The tropism of circulating tumor cells towards injured areas increases the risk of tumor recurrence. Recent studies have reported the inability of nanocarriers to penetrate the tumor site owing to short systemic half-life, high hydrostatic pressure, and inability to extravasate the tumor tissue. In this work, we developed simvastatin-loaded polycaprolactone - gelatin core-shell nanoparticles (SNP) for post-surgical management of breast cancer (BC). NP-loaded within the hyaluronic acid solution demonstrated shear thinning behavior (n = 0.3089). SNP demonstrated cytotoxic potential in the 2-D cell culture and 3-D tumoroids of MDA-MB-231 cell line in vitro. JC-1 and rhodamine-123 based assays demonstrated the ability of SNP to induce mitochondrial membrane damage. Further, cell cycle analysis revealed that the SNP prevented the progression of cell line from G0/G1 to S-phase. Simvastatin and SNP induced early apoptosis in the MDA-MB-231 cell line. Scratch, invasion, migration, and clonogenic assays demonstrated the ability of SNP to prevent cell invasion and clonal expansion. Immunocytochemistry revealed that both SIM and SNP reduced the expression of anti-apoptotic protein BCL-2 in metastatic cell line. Overall, the developed formulation demonstrates enormous potential for the post-surgery management of BC.

Liquid chromatography-high-resolution mass spectrometry-based metabolomics revealing the effects of zearalenone and alpha-zearalenol on human endometrial cancer cells.

Human exposure to mycotoxins through food involve a mixture of compounds, which can be harmful to human health. The Fusarium fungal species are known to produce zearalenone (ZEN), a non-steroidal estrogenic mycotoxin, and its metabolite alpha-zearalenol (α-ZEL), both of which possess endocrine-disruptive properties. Given their potential harm to human health through food exposure, investigating the combined effects of ZEN and α-ZEL becomes crucial. Hence, the combined impact of ZEN and α-ZEL study hold significant importance. This in vitro study delves into the critical area, examining their combined impact on the proliferation and metabolic profile of endometrial cancer Ishikawa cells via sulforhodamine, clonogenic, proliferating cell nuclear antigen (PCNA) and liquid chromatography-high resolution mass spectrometry (LC-HRMS) based untargeted metabolomics. Low concentrations of ZEN (25nm), α-ZEL (10nm), or a combination of both were observed to significantly enhance cell proliferation of Ishikawa cells, as evidenced by PCNA immunostaining, immunoblotting as well and clonogenic assays. The metabolomics revealed the perturbations in glycerophospholipid metabolism, nicotinate and nicotinamide metabolism and phenylalanine, tyrosine, tryptophan biosynthesis provides valuable insights into potential mechanism by which these mycotoxins may facilitate cell proliferation. However, further investigations are warranted to comprehensively understand the implications of these findings and their possible implications for human health.

MicroRNA-532-3p Modulates Colorectal Cancer Cell Proliferation and Invasion via Suppression of FOXM1.

Colorectal cancer (CRC) is a heterogeneous disease and classified into various subtypes, among which transcriptional alterations result in CRC progression, metastasis, and drug resistance. Forkhead-box M1 (FOXM1) is a proliferation-associated transcription factor which is overexpressed in CRC and the mechanisms of FOXM1 regulation have been under investigation. Previously, we showed that FOXM1 binds to promoters of certain microRNAs. Database mining led to several microRNAs that might interact with FOXM1 3'UTR. The interactions between shortlisted microRNAs and FOXM1 3'UTR were quantitated by a dual-luciferase reporter assay. MicroRNA-532-3p interacted with the 3'UTR of the FOXM1 mRNA transcript most efficiently. MicroRNA-532-3p was ectopically overexpressed in colorectal cancer (CRC) cell lines, leading to reduced transcript and protein levels of FOXM1 and cyclin B1, a direct transcriptional target of FOXM1. Further, a clonogenic assay was conducted in overexpressed miR-532-3p CRC cells that revealed a decline in the ability of cells to form colonies and a reduction in migratory and invading potential. These alterations were reinforced at molecular levels by the altered transcript and protein levels of the conventional EMT markers E-cadherin and vimentin. Overall, this study identifies the regulation of FOXM1 by microRNA-532-3p via its interaction with FOXM1 3'UTR, resulting in the suppression of proliferation, migration, and invasion, suggesting its role as a tumor suppressor in CRC.

First Dosimetric and Biological Verification for Spot-Scanning Hadron Arc Radiation Therapy With Carbon Ions

Purpose: Spot-scanning Hadron Arc therapy (SHArc) is a novel delivery technique for ion beams with potentially improved dose conformity and dose-averaged linear energy transfer (LETd) redistribution. The first dosimetric validation and in vitro verification of carbon ion arc delivery is presented. Methods: Intensity Modulated Particle Therapy (IMPT) and SHArc plans were designed to deliver homogenous physical dose or biological dose in a cylindrical PMMA phantom. Additional IMPT carbon plans were optimized for testing different LETd-boosting strategies. Verifications of planned doses were performed with an ionization chamber and clonogenic survival assay was conducted using A549 cancer lung cell line. Radiation-induced nuclear 53BP1 foci were assessed to evaluate the cellular response in both normoxic and hypoxic conditions. Results: Dosimetric measurements and clonogenic assay results showed a good agreement to planned dose and survival distributions. Measured survival fractions and foci confirmed carbon ions SHArc as potential modality to overcome hypoxia-induced radio-resistance. LETd-boosted IMPT plans reached similar LETd in the target as in SHArc plans promising similar features against hypoxia but at the cost of an increased entrance dose. SHArc resulted, however, in a lower dose bath but in a larger volume around the target. Conclusion: The first proof of principle of carbon ions SHArc delivery was performed and experimental evidence suggests this novel modality as an attractive approach for treating hypoxic tumor.

CMTM4 inhibits gastric tumorigenesis and metastasis.

CKLF-like MARVEL transmembrane domain-containing 4 (CMTM4) is involved in immune regulation and tumor progression; however, its role in gastric cancer (GC) remains unclear. This study explored the role and mechanism of CMTM4 in GC. Immunohistochemistry was used to analyze CMTM4 expression in human gastric biopsied cells from patients with GC (N=23) or chronic superficial gastritis (N=23). To investigate the function of CMTM4 in GC cells, the gene CMTM4 was knocked down and overexpressed in human gastric adenocarcinoma cell line AGS. The gene CMTM4 was overexpressed in AGS cells and human gastric cell line SGC7901. Cell Counting Kit 8 (CCK-8) and cell clonogenic assays were used to analyze the proliferation of the GC cells. Flow cytometry was used to analyze the effects of CMTM4 on apoptosis and the cell cycle. Wound healing and transwell assays were used to analyze the migration and invasion of the gastric cells, respectively. The mechanism of CMTM4 in GC cells was explored using the tandem mass tags (TMTs) proteome and verified by western blot analysis. CMTM4 expression was more downregulated in the human GC tissues than the gastritis tissues. CMTM4 overexpression significantly inhibited the proliferation, migration, and invasion of the GC cells, whereas CMTM4 knockdown enhanced gastric cell proliferation (P>0.05), migration (P>0.05), and invasion (P>0.05). Flow cytometry showed that CMTM4 promoted apoptosis and resulted in G1/S arrest in the GC cells. In addition, the proteome and western blot results showed that STAT1 was significantly upregulated, and the STAT1 signaling pathways were enriched in the GC cells overexpressing CMTM4. Our results suggest that CMTM4 plays a tumor-suppressive role in GC and may affect the growth, migration, and invasion of GC cells through the STAT1 signaling pathway. CMTM4 might have potential value as a prognosis marker and potential therapeutic target for GC therapy.

Cannabidiol exhibits potent anti-cancer activity against gemcitabine-resistant cholangiocarcinoma via ER-stress induction in vitro and in vivo

BackgroundFailure of treatment with gemcitabine in most cholangiocarcinoma (CCA) patients is due to drug resistance. The therapeutic potential of natural plant secondary compounds with minimal toxicity, such as cannabidiol (CBD), is a promising line of investigation in gemcitabine-resistant CCA. We aim to investigate the effects of CBD on gemcitabine-resistant CCA (KKU-213BGemR) cells in vitro and in vivo.MaterialsIn vitro, cell proliferation, colony formation, apoptosis and cell cycle arrest were assessed using MTT assay, clonogenicity assay and flow cytometry. The effect of CBD on ROS production was evaluated using the DCFH-DA fluorescent probe. The mechanism exerted by CBD on ER stress-associated apoptosis was investigated by western blot analysis. A gemcitabine-resistant CCA xenograft model was also used and the expression of PCNA and CHOP were evaluated by immunohistochemical analysis.ResultsThe IC50 values of CBD for KKU-213BGemR cells ranged from 19.66 to 21.05 µM. For a non-cancerous immortalized fibroblast cell line, relevant values were 18.29 to 19.21 µM. CBD suppressed colony formation by KKU-213BGemR cells in a dose-dependent manner in the range of 10 to 30 µM. CBD at 30 µM significantly increased apoptosis at early (16.37%) (P = 0.0024) and late (1.8%) stages (P < 0.0001), for a total of 18.17% apoptosis (P = 0.0017), in part by increasing ROS production (P < 0.0001). Multiphase cell cycle arrest significantly increased at G0/G1 with CBD 10 and 20 µM (P = 0.004 and P = 0.017), and at G2/M with CBD 30 µM (P = 0.005). CBD treatment resulted in increased expression of ER stress-associated apoptosis proteins, including p-PERK, BiP, ATF4, CHOP, BAX, and cytochrome c. In xenografted mouse, CBD significantly suppressed tumors at 10 and 40 mg/kg·Bw (P = 0.0007 and P = 0.0278, respectively), which was supported by an increase in CHOP, but a decrease in PCNA expression in tumor tissues (P < 0.0001).ConclusionThe results suggest that CBD exhibits potent anti-cancer activity against gemcitabine-resistant CCA in vitro and in vivo, in part via ER stress-mediated mechanisms. These results indicate that clinical explorative use of CBD on gemcitabine-resistant CCA patients is warranted.

Mutant P53 modulation by cryptolepine through cell cycle arrest and apoptosis in triple negative breast cancer

BackgroundTriple Negative Breast cancer is an aggressive breast cancer subtype. It has a more aggressive clinical course, an earlier age of onset, a larger propensity for metastasis, and worse clinical outcomes as evidenced by a higher risk of recurrence and a shorter survival rate. Currently, the primary options for TNBC treatment are surgery, radiation, and chemotherapy. These treatments however remain ineffective due to recurrence. However, given that p53 mutations have been identified in more than 60–88 % of TNBC, translating p53 into the clinical situation is particularly important in TNBC. In this study, we screened and evaluated the therapeutic potential of cryptolepine (CRP) in TNBC in-vitro models being an anti-malarial drug it could be repurposed as an anti-cancer therapeutic targeting TNBC. Moreover, the cytotoxicity activity of cryptolepine to TNBC cells and a detailed anti-tumor mechanism in mutant P53 has not been reported before. MethodsMTT assays were used to examine the cytotoxicity and cell viability activity of Cryptolepine in TNBC, non-TNBC T47D and MCF-7 and non-malignant MCF10A cells. Scratch wound and clonogenic assay was used to evaluate the cryptolepine’s effect on migration and colony forming ability of TNBC cells. Flow cytometry, MMP and DAPI was used to assess cell cycle arrest and cell apoptosis mechanism. The expression of proteins was detected by western blots. The differential expression of RNAs was evaluated by RT-PCR and the interaction between P53 and drug was evaluated computationally using in-silico approach and in-vitro using ChIP assay. ResultsIn this study, we found that cryptolepine has more preferential cytotoxicity in TNBC than non-TNBC cells. Notably, our studies revealed the mechanism by which cryptolepine induces intrinsic apoptosis and inhibit migration, colony formation ability, induce cell cycle arrest by inducing conformational change in the mutant P53 thereby increasing its DNA binding ability, hence activating its tumor suppressing potential significantly. ConclusionOur study revealed that CRP significantly reduced the proliferation, migration and colony forming ability of TNBC cells lines. Moreover, it was revealed that CRP induces cell cycle arrest and apoptosis by activating mutant P53 and enhancing its DNA binding ability to induce its tumor suppressing ability.

The Emission of Internal Conversion Electrons Rather Than Auger Electrons Increased the Nucleus-Absorbed Dose for 161Tb Compared with 177Lu with a Higher Dose Response for [161Tb]Tb-DOTA-LM3 Than for [161Tb]Tb-DOTATATE.

Preclinical data have shown that 161Tb-labeled peptides targeting the somatostatin receptor are therapeutically more effective for peptide receptor radionuclide therapy than are their 177Lu-labeled counterparts. To further substantiate this enhanced therapeutic effect, we performed cellular dosimetry to quantify the absorbed dose to the cell nucleus and compared dose-response curves to evaluate differences in relative biological effectiveness invitro. Methods: CA20948 cell survival was assessed after treatment with [161Tb]Tb- and [177Lu]Lu-DOTATATE (agonist) and with [161Tb]Tb- and [177Lu]Lu-DOTA-LM3 (antagonist) via a clonogenic assay. Cell binding, internalization, and dissociation assays were performed up to 7 d to acquire time-integrated activity coefficients. Separate S values for each type of particle emission (Auger/internal conversion [IC] electrons and β- particles) were computed via Monte Carlo simulations, while considering spheric cells. Once the absorbed dose to the cell nucleus was calculated, survival curves were fitted to the appropriate linear or linear-quadratic model and corresponding relative biological effectiveness was evaluated. Results: Although the radiopeptide uptake was independent of the radionuclide, [161Tb]Tb-DOTATATE and [161Tb]Tb-DOTA-LM3 delivered a 3.6 and 3.8 times higher dose to the nucleus, respectively, than their 177Lu-labeled counterparts on saturated receptor binding. This increased nucleus-absorbed dose was mainly due to the additional emission of IC and not Auger electrons by 161Tb. When activity concentrations were considered, both [161Tb]Tb-DOTATATE and [161Tb]Tb-DOTA-LM3 showed a lower survival fraction than did labeling with 177Lu. When the absorbed dose to the nucleus was considered, no significant difference could be observed between the dose-response curves for [161Tb]Tb- and [177Lu]Lu-DOTATATE. [161Tb]Tb-DOTA-LM3 showed a linear-quadratic dose response, whereas [161Tb]Tb-DOTATATE showed only a linear dose response within the observed dose range, suggesting additional cell membrane damage by Auger electrons. Conclusion: The IC, rather than Auger, electrons emitted by 161Tb resulted in a higher absorbed dose to the cell nucleus and lower clonogenic survival for [161Tb]Tb-DOTATATE and [161Tb]Tb-DOTA-LM3 than for the 177Lu-labeled analogs. In contrast, [161Tb]Tb-DOTATATE showed no higher dose response than [177Lu]Lu-DOTATATE, whereas for [161Tb]Tb-DOTA-LM3 an additional quadratic response was observed. Because of this quadratic response, potentially caused by cell membrane damage, [161Tb]Tb-DOTA-LM3 is a more effective radiopeptide than [161Tb]Tb-DOTATATE for labeling with 161Tb.