Clonogenic Survival Of Cells Research Articles

Differential CMS-Related Expression of Cell Surface Carbonic Anhydrases IX and XII in Colorectal Cancer Models-Implications for Therapy.

Tumor-associated carbonic anhydrases IX (CAIX) and XII (CAXII) have long been in the spotlight as potential new targets for anti-cancer therapy. Recently, CAIX/CAXII specific inhibitor SLC-0111 has passed clinical phase I study and showed differential response among patients with colorectal cancer (CRC). CRC can be classified into four different consensus molecular subgroups (CMS) showing unique expression patterns and molecular traits. We questioned whether there is a CMS-related CAIX/CAXII expression pattern in CRC predicting response. As such, we analyzed transcriptomic data of tumor samples for CA9/CA12 expression using Cancertool. Protein expression pattern was examined in preclinical models comprising cell lines, spheroids and xenograft tumors representing the CMS groups. Impact of CAIX/CAXII knockdown and SLC-0111 treatment was investigated in 2D and 3D cell culture. The transcriptomic data revealed a characteristic CMS-related CA9/CA12 expression pattern with pronounced co-expression of both CAs as a typical feature of CMS3 tumors. Protein expression in spheroid- and xenograft tumor tissue clearly differed, ranging from close to none (CMS1) to strong CAIX/CAXII co-expression in CMS3 models (HT29, LS174T). Accordingly, response to SLC-0111 analyzed in the spheroid model ranged from no (CMS1) to clear (CMS3), with moderate in CMS2 and mixed in CMS4. Furthermore, SLC-0111 positively affected impact of single and combined chemotherapeutic treatment of CMS3 spheroids. In addition, combined CAIX/CAXII knockdown and more effective treatment with SLC-0111 reduced clonogenic survival of CMS3 modelling single cells. In conclusion, the preclinical data support the clinical approach of targeted CAIX/CAXII inhibition by showing linkage of expression with response and suggest that patients with CMS3-classified tumors would most benefit from such treatment.

Improvement of Proton Irradiation Effectiveness of Human Lung Carcinoma Cells A 549 in the Presence of Gold Nanoparticles

Purpose: To study was to study the effect of GNP nanoparticles on tumor cells of human lung carcinoma A 549 when irradiated with protons.
 Materials and methods:
 Cell culture: Human lung carcinoma cells A 549.
 Gold nanoparticles Au/PEG 6000/W 200/30 nm: A colloidal solution of gold nanoparticles purchased from the firm M 9 Nanomaterials & Technologies was used in the work.
 Proton irradiation: The irradiation of cells was carried out on a therapeutic proton beam in the Medical and Technical Complex of the Laboratory of Nuclear Problems Joint Institute for Nuclear Research.
 Determination of radiosensitivity of cells: By determining the clonogenic survival of cells.
 Determination of the genotoxic activity of nanoparticles under the action of proton irradiation: investigated using a micronucleus test with blocking cytokinesis. The genotoxicity of gold nanoparticles was estimated by the number of micronuclei per 1000 binuclear cells. Micronucleus were counted only in binuclear cells. At least 1000 binuclear cells were calculated for each dose, experiments were carried out in three repeats.
 Results: The frequency of micronucleus formation indicates an increase in the genotoxic effect of nanoparticles when irradiated with protons at a dose of 2 Gy. Proton irradiation caused an increase in the frequency of micronucleus formation depending on the concentration of nanoparticles. When irradiated with protons at a dose of 2 Gy in the presence and absence of nanoparticles, the difference in the frequency of micronucleus formation for the concentration of nanoparticles was 2.5 mg/ml – 1.1; for 5 mg/ml and 10 mg/ml – 1.2; for 15 mg/ml – 1.3 and for 30 mg/ml –1.5. Cell survival curves reflect a decrease in their survival rate when metal nanoparticles with high Z are added, which reflects the occurrence of radiosensitization effects. The gain for 10 % and 50 % survival rates is 1.4 and 2.5, respectively.
 Conclusions: Under the influence of proton irradiation, the genotoxic activity of gold nanoparticles in human lung carcinoma cells A 549 increases, depending on their concentration. The survival rate of human lung carcinoma A 549 cells irradiated with protons in the presence of gold nanoparticles decreases.

Treatment of Orthotopic U251 Human Glioblastoma Multiforme Tumors in NRG Mice by Convection-Enhanced Delivery of Gold Nanoparticles Labeled with the β-Particle-Emitting Radionuclide, 177Lu.

In this study, we investigated convection-enhanced delivery (CED) of 23 ± 3 nm gold nanoparticles (AuNPs) labeled with the β-particle-emitting radionuclide 177Lu (177Lu-AuNPs) for treatment of orthotopic U251-Luc human glioblastoma multiforme (GBM) tumors in NRG mice. The cytotoxicity in vitro of 177Lu-AuNPs (0.0-2.0 MBq, 4 × 1011 AuNPs) on U251-Luc cells was also studied by a clonogenic survival assay, and DNA double-strand breaks (DSBs) caused by β-particle emissions of 177Lu were measured by confocal immunofluorescence microscopy for γH2AX. NRG mice with U251-Luc tumors in the right cerebral hemisphere of the brain were treated by CED of 1.1 ± 0.2 MBq of 177Lu-AuNPs (4 × 1011 AuNPs). Control mice received unlabeled AuNPs or normal saline. Tumor retention of 177Lu-AuNPs was assessed by single-photon emission computed tomography/computed tomography (SPECT/CT) imaging and biodistribution studies. Radiation doses were estimated for the tumor, brain, and other organs. The effectiveness for treating GBM tumors was determined by bioluminescence imaging (BLI) and T2-weighted magnetic resonance imaging (MRI) and by Kaplan-Meier median survival. Normal tissue toxicity was assessed by monitoring body weight and hematology and blood biochemistry analyses at 14 d post-treatment. 177Lu-AuNPs (2.0 MBq, 4 × 1011 AuNPs) decreased the clonogenic survival of U251-Luc cells to 0.005 ± 0.002 and increased DNA DSBs by 14.3-fold compared to cells treated with unlabeled AuNPs or normal saline. A high proportion of 177Lu-AuNPs was retained in the U251-Luc tumor in NRG mice up to 21 d with minimal re-distribution to the brain or other organs. The radiation dose in the tumor was high (599 Gy). The dose in the normal right cerebral hemisphere of the brain excluding the tumor was 93-fold lower (6.4 Gy), and 2000-3000-fold lower doses were calculated for the contralateral left cerebral hemisphere (0.3 Gy) or cerebellum (0.2 Gy). The doses in peripheral organs were <0.1 Gy. BLI revealed almost complete tumor growth arrest in mice treated with 177Lu-AuNPs, while tumors grew rapidly in control mice. MRI at 28 d post-treatment and histological staining showed no visible tumor in mice treated with 177Lu-AuNPs but large GBM tumors in control mice. All control mice reached a humane endpoint requiring sacrifice within 39 d (normal saline) or 45 d post-treatment (unlabeled AuNPs), while 5/8 mice treated with 177Lu-AuNPs survived up to 150 d. No normal tissue toxicity was observed in mice treated with 177Lu-AuNPs. We conclude that CED of 177Lu-AuNPs was highly effective for treating U251-Luc human GBM tumors in the brain in NRG mice at amounts that were non-toxic to normal tissues. These 177Lu-AuNPs administered by CED hold promise for treating patients with GBM to prevent recurrence and improve long-term outcome.

Abstract 3979: CRISPR/Cas9-mediated knockout of PLK4 results in antiproliferative response in human epidermoid carcinoma cells in vitro and in implanted xenografts

Abstract Non-melanoma skin cancers (NMSCs) constitute the largest number of cancers diagnosed in the United States, with an estimated 1 in 5 people affected by the age of 70. The most notable NMSCs are squamous cell carcinomas (SCCs) and basal cell carcinomas (BCCs), which are considered keratinocyte cancers due to their origin. Although the majority of NMSCs can be removed with surgery or curettage, these options are not always available if the tumor occurs in an inoperable location or becomes metastatic. While non-surgical treatment modalities are also currently available, they have not been fully effective in NMSC management. Therefore, novel mechanistic-based therapeutic strategies are needed. The polo-like kinases (PLKs) are a family of serine/threonine kinases that have been found to play multiple roles in regulating the cell cycle. PLK4 is a structurally unique member of this family, which has been shown to be essential for precise centriole duplication. However, PLK4 has been shown to be dysregulated in certain cancers. Though the role of PLK4 in NMSC is not fully understood, we previously showed that PLK4 was significantly overexpressed in NMSC cell lines at both mRNA and protein levels as compared to normal keratinocytes, and its small molecule inhibition resulted in anti-proliferative responses in NMSC cell lines (Cancer Res 2018; 78 [13 Suppl]: Abstract nr 547). Here, we further validated our previous observation by determining the effects of CRISPR/Cas9-mediated knockout (KO) of PLK4 in the A431 human SCC line, both in vitro and in vivo. Our data demonstrated significantly reduced cell growth of multiple A431 PLK4 KO clones compared to wild-type (WT) cells, as measured by RealTime-Glo MT cell viability and trypan blue exclusion assays. In addition, we found significant reduction in clonogenic survival of A431 PLK4 KO cells, as measured by colony formation assays. Using a Human Cancer Pathway Finder RT2 Profiler PCR array, we identified 18 of the 84 genes tested to be greater than 1.6-fold differentially regulated after PLK4 KO in these cells. Employing Ingenuity Pathway Analysis (IPA) software, we identified that the regulation of the epithelial-mesenchymal transition by growth factors was one of the top canonical pathways modulated by PLK4 KO. To determine the in vivo relevance of our in vitro data, we compared the tumorigenicity of the A431 WT and PLK4 KO cells in nu/nu mice. The mice (n=7 per group) were injected subcutaneously with 5 x 105 A431 WT or PLK4 KO cells and tumors were allowed to grow for further analyses. We found a significant reduction in tumor volume in A431 PLK4 KO xenografts as compared to A431 PLK4 WT xenografts. Taken together, these findings support our previous results that suggest PLK4 has pro-proliferative roles in NMSC and should be further studied as a potential novel target for skin cancer management. Citation Format: Mary A. Ndiaye, Debra R. Garvey, Gagan Chhabra, Chandra K. Singh, Nihal Ahmad. CRISPR/Cas9-mediated knockout of PLK4 results in antiproliferative response in human epidermoid carcinoma cells in vitro and in implanted xenografts [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 3979.

Abstract 5801: Xanthine oxidoreductase mediates the survival of starved lung adenocarcinoma cells by inducing UPR and autophagic degradation

Abstract Background &amp; Purpose: Lung adenocarcinoma (LUAD) is the most common and aggressive subtype of non-small cell lung cancer, and is the leading cause of cancer-associated mortality worldwide. Upregulation of purine degradation pathway is observed in LUAD. Xanthine oxidoreductase (XOR) is the rate-limiting enzyme in purine catabolism by converting hypoxanthine to xanthine and xanthine to uric acid. The altered expression and activity of XOR in cancer tissues are usually associated with prognosis of cancer, while its role in LUAD remains unknown. Methods: XOR expression in LUAD tissues were detected by immunohistochemistry. XOR knock-out LUAD cells were generated by CRISPR-Cas9 system. Clonogenic survival of LUAD cells was determined by crystal violet staining. The expression of genes involving in unfolded protein response (UPR) and autophagy was detected by RT-qPCR and the protein level was detected by Western blotting. Autophagic flux was observed by immunofluorescent microscopy. The level of amino acids and ATP/ADP/AMP were determined by UPLC-QTOFMS. Metabolic flux was analyzed by radio-labeled metabolites measured by UPLC-QTOFMS. Results: XOR is highly expressed in LUAD and significantly associated with poor clinical prognosis. Knocking out or inhibition of XOR attenuated the survival of starved LUAD cells. Nucleoside supplementation rescued the survival of starved LUAD cells upon XOR inhibition, while inhibition of purine nucleoside phosphorylase (PNP) impended the process, indicating that ribose produced by nucleoside degradation is required for the XOR-mediated the survival of LUAD cells. Accordingly, metabolic flux revealed that ribose derived from nucleoside fueled key carbon metabolic pathways to sustain the survival of starved LUAD cells. Mechanistically, downregulation of XOR suppressed UPR and autophagic degradation in starved LUAD cells. Consistently, inhibition of XOR decreased the level of amino acids produced by autophagic degradation, which was accompanied with down-regulation of mTORC1 signaling. Supplementation of amino acids including glutamine, glutamate or aspartate rescued the survival of starved LUAD cells, indicating that autophagic degradation plays a key role in XOR-mediated LUAD cells survival under nutrient stress. Finally, XOR inhibitors potentiated the anti-proliferative activity of 2-deoxy-D-glucose that induced UPR and/or autophagy in LUAD cells. Conclusions: We found that knock-out or inhibition of XOR attenuated UPR and autophagy in starved LUAD cells, which resulted in nutrients scarcity due to blockade of the degradation of nucleosides and proteins, and finally cell death. These findings demonstrate that XOR plays a crucial role in the survival of LUAD cells, suggesting that targeting XOR is a potential strategy to improve the efficacy of drugs that are able to induce UPR and autophagy. Citation Format: Manman Chen, Wei Guo, Xiaozhen Guo, Cen Xie, Linghua Meng. Xanthine oxidoreductase mediates the survival of starved lung adenocarcinoma cells by inducing UPR and autophagic degradation [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 5801.

Abstract 1839: Nelfinavir induced-cytotoxicity towards high-grade serous ovarian cancer cells involves induction of the unfolded protein response, modulation of protein synthesis, DNA damage, lysosomal impairment, and potentiation of toxicity caused by proteasome inhibition

Abstract High-grade serous ovarian cancer (HGSOC) is a significant cause of mortality among women worldwide. Traditional treatment consists of platinum-based therapy; however, the rapid development of platinum resistance contributes to lower life expectancy, warranting newer therapies to supplement the current platinum-based protocol. Repurposing market-available drugs as cancer therapeutics is a cost- and time-effective way to avail new therapies to drug-resistant patients. The anti-HIV agent nelfinavir (NFV) has shown promising toxicity against various cancers; however, its role against HGSOC is unknown. Here, we studied the effect of NFV against HGSOC cells obtained from patients along disease progression and carrying different sensitivities to platinum. NFV triggered, independently of platinum sensitivity, a dose-dependent reduction of HGSOC cell number and viability, and a parallel increase in hypo-diploid DNA content. Moreover, a dose-dependent reduction of clonogenic survival of cells escaping the acute toxicity was indicative of long-term residual damage. Western blot analysis of underlying molecular mechanisms revealed activation by NFV of the three signaling arms of the unfolded protein response (UPR): PERK, IRE1alpha and ATF6 - in a similar manner to the classical endoplasmic reticulum (ER) stressor tunicamycin. NFV-mediated modulation of the UPR was accompanied by the inhibition of global protein synthesis as analyzed through a non-radioactive method by labelling the nascent polypeptides with puromycin. NFV maintained a proapoptotic environment through the UPR, evidenced by a dose- and time-dependent enhanced expression of ATF4, CHOP, increased Bax/Bcl2 ratio, and cleavage of executioner caspase-7. Enhanced gamma-H2AX suggested NFV-mediated induction of DNA damage, which was associated with decreased proliferation signals driven by the AKT and ERK pathways. NFV increased the level of autophagosome-associated protein LC3II in HGSOC cells of varying platinum sensitivities; however, the autophagic flux was not increased during co-treatment with NFV and the lysosomal inhibitor bafilomycin A1, suggesting a likely impairment of the lysosomes by NFV impeding autophagosome clearance. Finally, we show that NFV potentiates the short-term cell cycle arrest and long-term toxicity caused by the proteasome inhibitor bortezomib. Overall, our in vitro study demonstrates that NFV can therapeutically target HGSOC cells of differential platinum sensitivities via several mechanisms, suggesting its prospective repurposing benefit considering its good safety profile. Citation Format: Mahbuba R. Subeha, Alicia A. Goyeneche, Prisca Bustamante, Michael A. Lisio, Julia V. Burnier, Carlos M. Telleria. Nelfinavir induced-cytotoxicity towards high-grade serous ovarian cancer cells involves induction of the unfolded protein response, modulation of protein synthesis, DNA damage, lysosomal impairment, and potentiation of toxicity caused by proteasome inhibition [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 1839.

Syntaxin 18 regulates the DNA damage response and epithelial-to-mesenchymal transition to promote radiation resistance of lung cancer

Radiotherapy is an important modality in lung cancer treatment. Despite advances in treatment planning and dose delivery, patient benefit is still limited by in-field relapse and metastatic recurrence. Simultaneous application of cisplatinum-based chemotherapy leads to moderately improved outcomes, thus providing proof-of-concept for radiosensitization strategies in lung cancer. In an unbiased functional genetic screen for radiosensitization targets in lung cancer, we identified syntaxin 18, a protein involved in retrograde vesicular transport between the Golgi apparatus and endoplasmic reticulum, as mediator of radioresistance. Downregulation of endogenous syntaxin 18 specifically reduced clonogenic survival of radioresistant and radiosensitive lung cancer cells following X-radiation. Gene expression programs regulating DNA repair, mitotic checkpoints and mitosis were altered in isogenic cells with reduced syntaxin 18 expression. Functionally, this translated into impaired DNA damage-induced cell cycle checkpoints leading to cell death by mitotic catastrophe. Interestingly, downregulation of syntaxin 18 in lung cancer cells also impaired expression of markers of epithelial-mesenchymal-transition, and reduced migration and invasion capacity. These findings suggest that syntaxin 18 is a key player regulating genes responsible for controlling the growth of the primary tumor as well as metastases upon radiotherapy of lung cancer. They provide a promising lead for biologically rational radiosensitization strategies impacting on radiation-induced cell death as well as metastasis.

Effective Treatment of Human Breast Carcinoma Xenografts with Single-Dose 211At-Labeled Anti-HER2 Single-Domain Antibody Fragment.

Single-domain antibody fragments (sdAbs) are attractive for targeted α-particle therapy, particularly with 211At, because of their rapid accumulation in tumor and clearance from normal tissues. Here, we evaluate the therapeutic potential of this strategy with 5F7 and VHH_1028-2 sdAbs that bind with high affinity to domain IV of human epidermal growth factor receptor type 2 (HER2). Methods: The HER2-specific sdAbs and HER2-irrelevant VHH_2001 were labeled using N-succinimidyl-3-211At-astato-5-guanidinomethyl benzoate (iso-211At-SAGMB). The cytotoxicity of iso- 211At-SAGMB-5F7 and iso- 211At-SAGMB-VHH_2001 were compared on HER2-expressing BT474 breast carcinoma cells. Three experiments in mice with subcutaneous BT474 xenografts were performed to evaluate the therapeutic effectiveness of single doses of iso- 211At-SAGMB-5F7 (0.7-3.0 MBq), iso- 211At-SAGMB-VHH_1028 (1.0-3.0 MBq), and iso- 211At-SAGMB-VHH_1028 and iso- 211At-SAGMB-VHH_2001 (∼1.0 MBq). Results: Clonogenic survival of BT474 cells was reduced after exposure to iso- 211At-SAGMB-5F7 (D0 = 1.313 kBq/mL) whereas iso- 211At-SAGMB-VHH_2001 was ineffective. Dose-dependent tumor growth inhibition was observed with 211At-labeled HER2-specific 5F7 and VHH_1028 but not with HER2-irrelevant VHH_2001. At the 3.0-MBq dose, complete tumor regression was seen in 3 of 4 mice treated with iso- 211At-SAGMB-5F7 and 8 of 11 mice treated with iso- 211At-SAGMB-VHH_1028; prolongation in median survival was 495% and 414%, respectively. Conclusion: Combining rapidly internalizing, high-affinity HER2-targeted sdAbs with the iso- 211At-SAGMB residualizing prosthetic agent is a promising strategy for targeted α-particle therapy of HER2-expressing cancers.

Evaluation of a Combination of an HDAC Inhibitor and a Cell Cycle Inhibitor in Pancreatic Cancer Cells

Pancreatic cancer is one of the deadliest cancers with 5‐year survival rate of ~ 10 percent. Late diagnosis, early metastasis, and resistance to therapeutics necessitate a better treatment approach for pancreatic cancer. Alterations in epigenetic profile through DNA and histone modifications enable tumor growth and metastasis. Evading the control of growth suppressors that regulate cell cycle is also a major hallmark of cancer pathophysiology. We aimed to study a combination therapeutic targeting histone deacetylase (HDAC) and key cell cycle regulator cyclin‐dependent kinase (CDK) in pancreatic cancer. We used panobinostat, a pan‐HDAC inhibitor approved for multiple myeloma and abemaciclib, an FDA‐approved CDK4/6 inhibitor for breast cancer. Our preliminary in vitro data in pancreatic cancer cells depicts synergistic cytotoxicity when treated in combination. In addition, combination treatment resulted in a significant decrease in proliferation and clonogenic survival of pancreatic cancer cells. Our mechanistic studies on the effects of this potent combination on histone modifications, gene expression, and cell cycle regulation will provide insights on drug synergy in pancreatic cancer cells. The results of our in vitro studies show that combining drugs that pharmacologically target two critical aspects of pancreatic cancer proliferation has a high potential for treating this lethal disease and as such merits further investigation.

Disruption of mitochondrial quality control genes promotes caspase-resistant cell survival following apoptotic stimuli

In cells undergoing cell-intrinsic apoptosis, mitochondrial outer membrane permeabilization (MOMP) typically marks an irreversible step in the cell death process. However, in some cases, a subpopulation of treated cells can exhibit a sublethal response, termed “minority MOMP.” In this phenomenon, the affected cells survive, despite a low level of caspase activation and subsequent limited activation of the endonuclease caspase-activated DNase (DNA fragmentation factor subunit beta). Consequently, these cells can experience DNA damage, increasing the probability of oncogenesis. However, little is known about the minority MOMP response. To discover genes that affect the MOMP response in individual cells, we conducted an imaging-based phenotypic siRNA screen. We identified multiple candidate genes whose downregulation increased the heterogeneity of MOMP within single cells, among which were genes related to mitochondrial dynamics and mitophagy that participate in the mitochondrial quality control (MQC) system. Furthermore, to test the hypothesis that functional MQC is important for reducing the frequency of minority MOMP, we developed an assay to measure the clonogenic survival of caspase-engaged cells. We found that cells deficient in various MQC genes were indeed prone to aberrant post-MOMP survival. Our data highlight the important role of proteins involved in mitochondrial dynamics and mitophagy in preventing apoptotic dysregulation and oncogenesis.

Nelfinavir Induces Cytotoxicity towards High-Grade Serous Ovarian Cancer Cells, Involving Induction of the Unfolded Protein Response, Modulation of Protein Synthesis, DNA Damage, Lysosomal Impairment, and Potentiation of Toxicity Caused by Proteasome Inhibition.

Simple SummaryHigh-grade serous ovarian cancer (HGSOC) accounts for 70% of all ovarian-cancer-related deaths. Mainstay treatment with platinum-based drugs following surgery results in favorable outcomes in the majority of patients; however, in >80% of cases, the disease relapses with eventual drug resistance. As such, urgent development of improved alternative therapies is necessary for HGSOC patients with lower life expectancy. Rapid repurposing of market available drugs for cancer therapy is a cost-effective alternative to bypass the decade-long traditional drug development pipeline. Among potential drug-repurposing candidates, nelfinavir (NFV)—an anti-infective agent to treat acquired immunodeficiency syndrome (AIDS)—has shown anti-cancer effects against diverse cancers; however, its remedial benefits against HGSOC are unknown. In this study, we explored how NFV targets HGSOC cells obtained from patients at platinum-sensitive and -resistant stages. We observed beneficial efficacy elicited by NFV against HGSOC in both disease conditions through multiple mechanistic avenues, suggesting positive drug-repurposing prospects.High-grade serous ovarian cancer (HGSOC) is a significant cause of mortality among women worldwide. Traditional treatment consists of platinum-based therapy; however, rapid development of platinum resistance contributes to lower life expectancy, warranting newer therapies to supplement the current platinum-based protocol. Repurposing market-available drugs as cancer therapeutics is a cost- and time-effective way to avail new therapies to drug-resistant patients. The anti-HIV agent nelfinavir (NFV) has shown promising toxicity against various cancers; however, its role against HGSOC is unknown. Here, we studied the effect of NFV against HGSOC cells obtained from patients along disease progression and carrying different sensitivities to platinum. NFV triggered, independently of platinum sensitivity, a dose-dependent reduction in the HGSOC cell number and viability, and a parallel increase in hypo-diploid DNA content. Moreover, a dose-dependent reduction in clonogenic survival of cells escaping the acute toxicity was indicative of long-term residual damage. In addition, dose- and time-dependent phosphorylation of H2AX indicated NFV-mediated DNA damage, which was associated with decreased survival and proliferation signals driven by the AKT and ERK pathways. NFV also mediated a dose-dependent increase in endoplasmic reticulum stress-related molecules associated with long-term inhibition of protein synthesis and concurrent cell death; such events were accompanied by a proapoptotic environment, signaled by increased phospho-eIF2α, ATF4, and CHOP, increased Bax/Bcl-2 ratio, and cleaved executer caspase-7. Finally, we show that NFV potentiates the short-term cell cycle arrest and long-term toxicity caused by the proteasome inhibitor bortezomib. Overall, our in vitro study demonstrates that NFV can therapeutically target HGSOC cells of differential platinum sensitivities via several mechanisms, suggesting its prospective repurposing benefit considering its good safety profile.

DUSP6 regulates radiosensitivity in glioblastoma by modulating the recruitment of phosphorylated DNAPKcs at DNA double-strand breaks.

Glioblastoma (GBM) has poor median survival due to its resistance to chemoradiotherapy, which results in tumor recurrence. Recurrent GBMs currently lack effective treatments. DUSP6 is known to be pro-tumorigenic and is upregulated in GBM. We show that DUSP6 expression is significantly higher in recurrent GBM patient biopsies compared to expression levels in primary GBM biopsies. Importantly, although it has been reported to be a cytoplasmic protein, we found nuclear localization of DUSP6 in primary and recurrent patient samples and in parent and relapse populations of GBM cell lines generated from an in vitro radiation survival model. DUSP6 inhibition using BCI resulted in decreased proliferation and clonogenic survival of parent and relapse cells. Pharmacological or genetic inhibition of DUSP6 catalytic activity radiosensitized primary and, importantly, relapse GBM cells by inhibiting the recruitment of phosphorylated DNAPKcs (also known as PRKDC), subsequently downregulating the recruitment of phosphorylated histone H2AX (γH2AX) and 53BP1 (also known as TP53BP1). This resulted in decreased cell survival and prolonged growth arrest upon irradiation in vitro and significantly increased the progression-free survival in orthotopic mouse models of GBM. Our study highlights a non-canonical function of DUSP6, emphasizing the potential application of DUSP6 inhibitors in the treatment of recurrent GBM.

Oligonucleotide-Functionalized Gold Nanoparticles for Synchronous Telomerase Inhibition, Radiosensitization, and Delivery of Theranostic Radionuclides.

Telomerase represents an attractive target in oncology as it is expressed in cancer but not in normal tissues. The oligonucleotide inhibitors of telomerase represent a promising anticancer strategy, although poor cellular uptake can restrict their efficacy. In this study, gold nanoparticles (AuNPs) were used to enhance oligonucleotide uptake. “match” oligonucleotides complementary to the telomerase RNA template subunit (hTR) and “scramble” (control) oligonucleotides were conjugated to diethylenetriamine pentaacetate (DTPA) for 111In-labeling. AuNPs (15.5 nm) were decorated with a monofunctional layer of oligonucleotides (ON–AuNP) or a multifunctional layer of oligonucleotides, PEG(polethylene glycol)800-SH (to reduce AuNP aggregation) and the cell-penetrating peptide Tat (ON–AuNP–Tat). Match–AuNP enhanced the cellular uptake of radiolabeled oligonucleotides while retaining the ability to inhibit telomerase activity. The addition of Tat to AuNPs increased nuclear localization. 111In–Match–AuNP–Tat induced DNA double-strand breaks and caused a dose-dependent reduction in clonogenic survival of telomerase-positive cells but not telomerase-negative cells. hTR inhibition has been reported to sensitize cancer cells to ionizing radiation, and 111In–Match–AuNP–Tat therefore holds promise as a vector for delivery of radionuclides into cancer cells while simultaneously sensitizing them to the effects of the emitted radiation.

DNA-PKc inhibition overcomes taxane resistance by promoting taxane-induced DNA damage in prostate cancer cells.

Overcoming taxane resistance remains a major clinical challenge in metastatic castrate-resistant prostate cancer (mCRPC). Loss of DNA repair proteins is associated with resistance to anti-microtubule agents. We propose that alterations in DNA damage response (DDR) pathway contribute to taxane resistance, and identification of these alterations may provide a potential therapeutic target to resensitize docetaxel-refractory mCRPC to taxane-based therapy. Alterations in DDR gene expression in our prostate cancer cell line model of docetaxel-resistance (DU145-DxR) derived from DU-145 cells were determined by DDR pathway-specific polymerase chain reaction array and immunoblotting. The PRKDC gene encoding DNA-PKc (DNA-dependent protein kinase catalytic unit), was noted to be overexpressed and evaluated for its role in docetaxel resistance. Cell viability and clonogenic survival of docetaxel-treated DU145-DxR cells were assessed after pharmacologic inhibition of DNA-PKc with three different inhibitors-NU7441, LTURM34, and M3814. Response to second-line cytotoxic agents, cabazitaxel and etoposide upon DNA-PKc inhibition was also tested. The impact of DNA-PKc upregulation on DNA damage repair was evaluated by comet assay and analysis of double-strand breaks marker, γH2AX and Rad51. Lastly, DNA-PKc inhibitor's effect on MDR1 activity was assessed by rhodamine 123 efflux assay. DDR pathway-specific gene profiling revealed significant upregulation of PRKDC and CDK7, and downregulation of MSH3 in DU145-DxR cells. Compared to parental DU145, DU145-DxR cells sustained significantly less DNA damage when exposed to etoposide and docetaxel. Pharmacologic inhibition of DNA-PKc, a component of NHEJ repair machinery, with all three inhibitors, significantly resensitized DU145-DxR cells to docetaxel. Furthermore, DNA-PKc inhibition also resensitized DU145-DxR to cabazitaxel and etoposide, which demonstrated cross-resistance. Inhibition of DNA-PKc led to increased DNA damage in etoposide- and docetaxel-treated DU145-DxR cells. Finally, DNA-PKc inhibition did not affect MDR1 activity, indicating that DNA-PKc inhibitors resensitized taxane-resistant cells via an MDR1-independent mechanism. This study supports a role of DDR genes, particularly, DNA-PKc in promoting resistance to taxanes in mCRPC. Targeting prostatic DNA-PKc may provide a novel strategy to restore taxane sensitivity in taxane-refractory mCRPC.

RBM5-AS1 promotes radioresistance in medulloblastoma through stabilization of SIRT6 protein

Cancer stem cells (CSCs) contribute to radioresistance in medulloblastoma. Thus, identification of key regulators of medulloblastoma stemness is critical for improving radiotherapy for medulloblastoma. In the present study, we profiled CSC-related long non-coding RNAs (lncRNAs) between radioresistant and parental medulloblastoma cells. The roles of the lncRNA RBM5-AS1 in the stemness and radiosensitivity of medulloblastoma cells were investigated. We found that RBM5-AS1, a novel inducer of medulloblastoma stemness, was significantly upregulated in radioresistant medulloblastoma cells compared to parental cells. Knockdown of RBM5-AS1 diminished the viability and clonogenic survival of both radioresistant and parental medulloblastoma cells after radiation. Silencing of RBM5-AS1 significantly enhanced radiation-induced apoptosis and DNA damage. In vivo studies confirmed that depletion of RBM5-AS1 inhibited tumor growth and increased radiosensitivity in a medulloblastoma xenograft model. In contrast, overexpression of RBM5-AS1 reduced radiation-induced apoptosis and DNA damage in medulloblastoma cells. Mechanistically, RBM5-AS1 interacted with and stabilized sirtuin 6 (SIRT6) protein. Silencing of SIRT6 reduced the stemness and reinforced radiation-induced DNA damage in medulloblastoma cells. Overexpression of SIRT6 rescued medulloblastoma cells from RBM5-AS1 depletion-induced radiosensitization and DNA damage. Overall, we identify RBM5-AS1 as an inducer of stemness and radioresistance in medulloblastoma. Targeting RBM5-AS1 may represent a potential strategy to overcome the resistance to radiotherapy in this malignancy.

Repurposing an Antiepileptic Drug for the Treatment of Glioblastoma

Glioblastoma multiforme (GBM) is a grade IV malignant glioma. It is a highly proliferative brain tumor with the 5-year survival rate close to 5% indicating a high mortality rate. Current treatment strategies include surgery, radiation, and chemotherapy. Development of resistance against the current treatment options and the blood brain barrier (BBB) pose major challenges in GBM management. Therefore, we studied an FDA-approved drug stiripentol which is used for Dravet's syndrome (a rare form of epilepsy) for its efficacy in GBM. Stiripentol decreased the cytotoxicity in U87 and U138 astrocytoma cells. Cell proliferation and clonogenic survival of U87 cells was also adversely affected by stiripentol. Cellular anticancer activity was studied by cell cycle assay, apoptosis assay by flow cytometry, and by measuring the expression of apoptotic markers by Western blotting. In addition, effect of stiripentol on key cell signaling proteins was determined using Western blotting. Stiripentol treatment induced apoptosis and resulted in cell cycle arrest in GBM cells. 3D spheroid assay was performed to replicate the tumor environment and analyze the multidirectional metastatic growth of the cancer cells in presence of stiripentol and the standard GBM therapeutic Temozolomide (TMZ). Fluorescence imaging of spheroids demonstrated anticancer effects of stiripentol and TMZ. Further in vivo studies will be performed on xenograft models of astrocytoma cells. Our results indicate that stiripentol can be an effective GBM therapeutic and merits further investigation.

Manganese Ferrite Nanoparticles Enhance the Sensitivity of Hepa1-6 Hepatocellular Carcinoma to Radiation by Remodeling Tumor Microenvironments.

We evaluated the effect of manganese ferrite nanoparticles (MFN) on radiosensitization and immunologic responses using the murine hepatoma cell line Hepa1-6 and the syngeneic mouse model. The clonogenic survival of Hepa1-6 cells was increased by hypoxia, while being restricted by ionizing radiation (IR) and/or MFN. Although MFN suppressed HIF-1α under hypoxia, the combination of IR and MFN enhanced apoptosis and DNA damage in Hepa1-6 cells. In the Hepa1-6 syngeneic mouse model, the combination of IR and MFN notably limited the tumor growth compared to the single treatment with IR or MFN, and also triggered more frequent apoptosis in tumor tissues than that observed under other conditions. Increased expression of PD-L1 after IR was not observed with MFN alone or the combination of IR and MFN in vitro and in vivo, and the percentage of tumor-infiltrating T cells and cytotoxic T cells increased with MFN, regardless of IR, in the Hepa1-6 syngeneic mouse model, while IR alone led to T cell depletion. MFN might have the potential to overcome radioresistance by alleviating hypoxia and strengthening antitumor immunity in the tumor microenvironment.

Depletion of Survivin suppresses docetaxel-induced apoptosis in HeLa cells by facilitating mitotic slippage

The anticancer effects of taxanes are attributed to the induction of mitotic arrest through activation of the spindle assembly checkpoint. Cell death following extended mitotic arrest is mediated by the intrinsic apoptosis pathway. Accordingly, factors that influence the robustness of mitotic arrest or disrupt the apoptotic machinery confer drug resistance. Survivin is an inhibitor of apoptosis protein. Its overexpression is associated with chemoresistance, and its targeting leads to drug sensitization. However, Survivin also acts specifically in the spindle assembly checkpoint response to taxanes. Hence, the failure of Survivin-depleted cells to arrest in mitosis may lead to taxane resistance. Here we show that Survivin depletion protects HeLa cells against docetaxel-induced apoptosis by facilitating mitotic slippage. However, Survivin depletion does not promote clonogenic survival of tumor cells but increases the level of cellular senescence induced by docetaxel. Moreover, lentiviral overexpression of Survivin does not provide protection against docetaxel or cisplatin treatment, in contrast to the anti-apoptotic Bcl-xL or Bcl-2. Our findings suggest that targeting Survivin may influence the cell response to docetaxel by driving the cells through aberrant mitotic progression, rather than directly sensitizing cells to apoptosis.

Polydatin Induces Differentiation and Radiation Sensitivity in Human Osteosarcoma Cells and Parallel Secretion through Lipid Metabolite Secretion.

Osteosarcoma is a bone cancer characterized by the production of osteoid tissue and immature bone from mesenchymal cells. Osteosarcoma mainly affects long bones (femur is most frequently site) and occur in children and young adults with greater incidence. Here, we investigated the role accomplished by polydatin, a natural antioxidative compound, in promoting osteogenic differentiation alone or after radiation therapy on osteosarcoma cells. In vitro, polydatin significantly induced cell cycle arrest in S-phase and enhanced bone alkaline phosphatase activity. Moreover, the differentiation process was paralleled by the activation of Wnt-β-catenin pathway. In combination with radiotherapy, the pretreatment with polydatin promoted a radiosensitizing effect on osteosarcoma cancer cells as demonstrated by the upregulation of osteogenic markers and reduced clonogenic survival of tumor cells. Additionally, we analyzed, by mass spectrometry, the secretion of sphingolipid, ceramides, and their metabolites in osteosarcoma cells treated with polydatin. Overall, our results demonstrate that polydatin, through the secretion of sphingolipids and ceramide, induced osteogenic differentiation, alone and in the presence of ionizing therapy. Future investigations are needed to validate the use of polydatin in clinical practice as a potentiating agent of radiotherapy-induced anticancer effects.

The secretome of non-tumorigenic mammary cells MCF-10A elicits DNA damage in MCF-7 and MDA-MB-231 breast cancer cells

Radiotherapy is used in breast cancer to destroy tumor cells lingering after surgery. It is accepted that lethal effects of ionizing radiation occur as a result of damage to DNA in irradiated (IR) cells. However, response mechanisms may promote cell survival with efficient DNA repair or genomic alterations. Chromosomal aberrations are frequent in surviving cells and may enhance chromosomal instability (CIN) which is associated with increased risk of recurrence and metastasis. Intercellular communication can affect the response in IR cells and cause damage in non-irradiated (N-IR) cells. We evaluated the effect of the secretome of non-tumorigenic mammary cells (MCF-10A) on proliferation and DNA damage in breast cancer cells (MCF-7 and MDA-MB-231). Results showed that conditioned media from IR and N-IR MCF-10A cells produced cycles of DNA double-strand breaks in N-IR and IR tumor cells leaving them with residual damage. CIN markers (micronuclei, nucleoplasmic bridges, nuclear buds) were also increased in IR and N-IR tumor cells, being the effect of conditioned media from IR MCF-10A greater in many cases. The inhibition of phosphorylation/activation of Src kinase in cancer cells hindered CIN markers' increment. Besides, clonogenic survival of tumor cells was differentially modulated by conditioned media from MCF-10A: decreased in MCF-7 and enhanced in MDA-MB-231 cells. These results signal the relevance of tumor-host interaction in tumor behavior and the response to radiotherapy.