Synergistic Cytotoxicity Research Articles

Abstract 954: PIK3CA mutation induces immunogenicity and increases the immune checkpoint inhibitor response in urothelial carcinoma

Abstract Background: Immune checkpoint inhibitor (ICI) has been widely used for advanced urothelial carcinoma (mUC) globally. However, little is known about the potential predictive factors of the ICI response. The study aims to identify a potential biomarker to predict ICI response in UC. Material and methods: We performed targeted next generation sequencing (NGS) of pre-treatment tumor Formalin-fixed paraffin-embedded (FFPE) samples by using ACTOnco® comprehensive genomic panel (ACT Genomics, Co. Ltd., Taiwan) to target all coding exons of 440 cancer-associated genes. We retrospective analyzed a total of 70 patients with mUC underwent at least one cycle of ICI with or without chemotherapy at Kaohsiung Chang Gung Memorial Hospital between April 2016 and November 2019. Patients were defined as responders (CR/PR) and non-responders (SD/PD) according to the RECIST evaluation. TMB was estimated by calculating all coding exons variants, including somatic non-synonymous, frameshift and splice site variants. Results: A total of 55 UC tumor samples passed QC process were analyzed. The median age was 67, and 74% of all patients were in ICI monotherapy cohort. The most common genetic alterations were TP53 (66%), KMT2D (42%) and MUC16 (32%). In ICI monotherapy group, responders had a higher TMB than non-responders (median 19.5 vs 6.5 mt/Mb; p = 0.004). Patients with high TMB on ICI monotherapy had a significantly longer PFS (14.6 vs 2.9 months; p = 0.003) and OS (NR vs 11.0 months; p = 0.01) than TMB low patients. We found that PIK3CA (32% vs 3%), UBR5 (23% vs 0%), PRKDC (32% vs 10%) were the top 3 enriched mutations between responders and non-responders. All PIK3CA mutation were hot-spot mutation, and tumor harbored PIK3CA mutation had a median high TMB than PIK3CA wild type in UC, NSCLC, breast and colorectal cancer. By analyzing tumor and immune microenvironment by TISIDB platform, UC with PIK3CA mutation was highly associated with increasing MHC molecules expression (HLA-A, HLA-B, HLA-C, B2M) in UC and gastric cancer. In vitro study, we confirmed anti-PDL1 with PBMC had a synergistic cytotoxicity in PIK3CA mutated cell lines (BFTC-909, AGS and DLD-1), but not in PIK3CA wild type cell lines (T24, RT4, UMUC-14, N87 and SW837). Knocking-down PIK3CA expression diminished the cytotoxic activity of anti-PDL1 with PBMC in BFTC-909 and AGS cells. PIK3CA down-regulation inhibits antigen presentation machinery process. We confirmed that knockout PIK3CA gene function by CRISPR/Cas9 decreased MHC1 and B2M molecules expression, and showing modest resistance to ICI than mice with preserved PIK3CA function. Conclusion: UC with PIK3CA missense mutation exhibits a higher immunogenicity through enhancing MHC class I and B2M expression. PIK3CA missense mutation is a potential predictive marker for ICI response in UC. Citation Format: Harvey Yu-Li Su, Ling-Yi Xiao, Shih-Yu Huang, Li-Chung Chang, Yi-Hua Chen, Hao-Lun Luo, Chia-Ling Wu. PIK3CA mutation induces immunogenicity and increases the immune checkpoint inhibitor response in urothelial carcinoma [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 954.

Abstract 286: Targeting metabolic vulnerabilities in acute myeloid leukemia

Abstract Glutamine (Gln) is the most abundant amino acid intracellularly and in the plasma. Importantly, Gln depletion has emerged as a therapeutic approach for cancers that have undergone metabolic reprogramming and have exhibited a dependence on Gln for survival, including acute myeloid leukemia (AML). Asparaginases are bacterial enzymes that hydrolyze plasma glutamine to glutamate and ammonia, depriving glutamine-dependent AML cells from one of their vital nutrients. Preliminary data and literature have shown that following treatment with asparaginases in vitro there is a consistent increase in serine (Ser). Therefore, we hypothesized that in response to Gln depletion, AML cells upregulate de novo Ser biosynthesis as a compensatory pathway, and the inhibition of this pathway will synergize with Gln depletion to increase cytotoxicity in AML cells. We will test this by targeting, phosphoglycerate dehydrogenase (PHGDH), a key enzyme in Ser biosynthesis, which is a very common target for serine biosynthesis. We determined that crisantaspase (Rylaze) inhibited the proliferation of several AML cell lines with pharmacologically relevant IC50 values. Similarly, decreasing the concentration of Gln in the media resulted in a dose-dependent decrease in the proliferation, growth, and expansion of AML cells, confirming AML sensitivity to Gln depletion. Protein expression of PHGDH and PSAT was upregulated following Gln withdrawal from the media as well as following crisantaspase treatment. We set out to explore the effects of PHGDH knockout and observed that the PHGDH knockout AML cells were significantly more sensitive to crisantaspase, yielding IC50 values ranging from, ~250-fold lower compared to the parental lines. Gln depletion through crisantaspase treatment or withdrawal from the media decreased GSH levels and increased LDH release, both of which were further enhanced by PHGDH knockout. Indicating enhanced oxidative stress and cytotoxicity when both Gln and Ser are targeting in combination. Additionally, silencing of PHGDH reinforced anti-leukemic effects of glutamine modulation by different glutamine metabolism inhibitors, including V9302, CB-839 and BPTES. Preliminary testing of an NAD-dependent cell permeable PHGDH competitive inhibitor prodrug (BI4916) demonstrated an inhibition of AML cell proliferation in multiple cell lines. It has also demonstrated potential synergism and increase in cellular cytotoxicity when used in combination with Rylaze. Simultaneous targeting of Ser and Gln metabolism for AML treatment shows promising results in vitro with great potential for translation into clinical practice with clinically available asparaginases, and other glutamine modulators. Ongoing work is focused on the design of a more potent pharmacologic inhibitor of PHGDH to combine with crisantaspase as a novel treatment approach for AML. Citation Format: Kanwal Mahmood Hameed, Ashkan Emadi. Targeting metabolic vulnerabilities in acute myeloid leukemia [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 286.

Abstract 6123: Characterization of the synergistic tumor cytotoxicity of agonistic DR5 IgM antibody IGM-8444 with chemotherapeutic agents

Abstract Death receptor 5 (DR5) is a tumor necrosis factor receptor (TNF) superfamily member that requires multimerization to activate the extrinsic apoptotic pathway and is broadly expressed on solid and hematologic cancers. IGM-8444 is a multivalent IgM DR5 agonist that efficiently multimerizes DR5 to induce tumor cell apoptosis while maintaining a favorable in vitro and in vivo safety profile. The universal mechanism of apoptotic cell death and the safety profile of IGM-8444 makes it an attractive combination partner with standard of care treatment regimens. Here we describe the characterization of single agent and combinatorial cytotoxicity with different classes of chemotherapeutic agents. IGM-8444 was first evaluated as a monotherapy across a panel of human solid tumor cell lines in vitro and xenograft tumor models in vivo. IGM-8444 responses ranged from highly sensitive to resistant. In a sensitive Colo205 model IGM-8444 additionally showed rapid intratumoral pharmacodynamic (PD) activity, inducing maximal caspase-3 cleavage at 6 hours post-dose. Next a panel of solid tumor (including colorectal, gastric, non-small cell lung cancer, and pancreatic) cell lines were selected to evaluate IGM-8444 in combination with standard of care chemotherapeutic agents. Synergistic cytotoxicity was observed when IGM-8444 was used in combination with certain classes of chemotherapeutic agents including topoisomerase inhibitors, microtubule inhibitors, nucleoside analogs, and platinum-based agents. Enhanced anti-tumor activity was also observed in xenograft mouse models where IGM-8444 was dosed in combination with these classes of chemotherapeutic compounds. Mechanistically, this synergistic combinatorial cytotoxicity may be explained by the reported increase in DR5 expression on tumor cells following treatment with many of these classes of chemotherapeutic agents. In summary, IGM-8444 shows in vitro cytotoxicity and in vivo PD and anti-tumor efficacy responses in preclinical models, with enhanced activity in combination with several classes of chemotherapies reported to upregulate DR5 expression. IGM-8444 combination with FOLFIRI standard of care is currently under evaluation in a Phase 1 study in patients with metastatic colorectal cancer (NCT04553692). Citation Format: Beatrice T. Wang, Thomas J. Matthew, Poonam Yakkundi, Miho Oyasu, Mélanie Desbois, Susan E. Calhoun, Ling Wang, Tasnim Kothambawala, Devinder K. Ubhi, Marvin S. Peterson, Eric W. Humke, Maya F. Kotturi, Bruce A. Keyt, Angus M. Sinclair. Characterization of the synergistic tumor cytotoxicity of agonistic DR5 IgM antibody IGM-8444 with chemotherapeutic agents [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 6123.

Abstract 4777: Exploring ferroptosis pathway for the development of therapeutic strategy in liposarcoma

Abstract Background: Liposarcoma (LPS) is one of the most common soft tissue sarcoma (STS) subtypes. Both well-differentiated LPS (WDLPS) and dedifferentiated LPS (DDLPS) have 12q13-15 amplification. Mouse double minute 2 homolog (MDM2) is one of the most important oncogenes within this region. Ferroptosis is a unique type of necrotic cell death featured accumulation of lipid-based reactive oxygen species (ROS) derived from the oxidative modification of phospholipid membranes. Cysteine metabolism is the critical part of ferroptosis regulation. Cystine-glutamate antiporter (system xc-; xCT), encoded by two subcomponents, the solute carrier family 7 member 11 (SLC7A11) and SLC3A2, is responsible for cystine supply from extra-cellular environment. Imported cystine is then reduced to cysteine, a key component of tripeptide glutathione (GSH). Glutathione peroxidase 4 (GPX4) requires GSH to repair lipid peroxidation and prevent ferroptosis. Inactivation of GPX4, either through cystine deprivation due to xCT inhibitor erastin, or by GPX4 inhibitor Ras-selective lethal small molecule 3 (RSL3), will lead to accumulation of lipid-based ROS, and eventually ferroptosis cell death. The tumor suppressor p53 (TP53) could suppress the expression of SLC7A11 and induce ferroptosis. TP53 could also indirectly suppress 3-hydroxy-3-methyl-glutaryl (HMG)-coenzyme A (CoA) reductase (HMGCR) activity, a key pathway for generation of biomolecules with anti-ferroptosis property. MDM2, the key oncogene in DDLPS, and homolog MDM4 could facilitate ferroptotic death in TP53-dependent or -independent mechanisms. Methods: We explored public domain database to identify possible aberrations of ferroptosis-related genes in DDLPS. We then examined the sensitivity of DDLPS cell lines to possible ferroptosis-inducing agents. Furthermore, we used nutlin-3, a MDM2 inhibitor, to modulate the MDM2 expression in DDLPS cell line, and explored the potential synergistic effect of nutlin-3 with ferroptosis-inducing agents. Finally, we used immunoblotting study to reveal the potential mechanism responsible to possible synergistic effect. Results: Expression level of GPX4 is significantly lower in DDLPS than adipose tissue and WDLPS, and DPP4 was general higher in LPS than adipose tissue. HMGCR, SLC7A11 and SLC3A2 were mostly up-regulated in LPS in comparison with benign counterpart. Both DDLPS cell lines showed sensitivity to erastin and RSL3. In addition, nutlin-3 could exert synergistic ferroptosis-inducing effect and cytotoxicity with erastin and RSL3 in sequential manner. Furthermore, nultin-3 treatment could upregulate SLC3A2 with altered cystine/glutamate exchange in DDLPS cell lines, indicating possible mechanism responsible for nultin-3 resistance and the synergistic effect of nultin-3 with erastin or RSL3. Conclusion: Our study showed that modulation of ferroptosis is a potential treatment strategy in DDLPS. Citation Format: Chueh-Chuan Yen, San-Chi Chen, Chih-Hsueh Chen, Jir-You Wang, Chao-Ming Chen, Po-Kuei Wu, Muh-Hwa Yang. Exploring ferroptosis pathway for the development of therapeutic strategy in liposarcoma. [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 4777.

Abstract 2937: Preclinical characterization of CBA-1535, a novel bi-specific tribody, with two binding sites to 5T4 and one site to CD3ε

Abstract 5T4 oncofetal tumor-associated antigen (also known as TPGB or WAIF1) is over-expressed in a wide range of solid tumors but shows very limited expression in normal adult tissues thus suggesting 5T4 as an attractive target for antibody therapy. CBA-1535, originally developed by Biotecnol (UK), is a novel trivalent biological construct named a Tribody™ with two binding sites to the tumor associated antigen 5T4 and one to the T cell expressed antigen CD3ε. CD-3 based T cell engagers (TCE) is one of the attractive strategy to treat cancer patients resistant to conventional therapies, however, the effects of TCE on solid tumors are limited due to the immunosuppressive tumor microenvironment. To overcome this limitation, CBA-1535 is designed to have stronger binding activity to 5T4 with two binding sites, and to have the lower affinity to CD3 than to 5T4 to prevent non-specific T cell activation.CBA-1535 does not have a Fc region with a molecular weight of ~100 kDa and it has a low nM Kd for bivalent binding to 5T4, a 20 to 30 nM Kd for monovalent binding to CD3ε. CBA-1535 efficiently induced T cell activation in human PBMCs as exemplified as CD25 and CD69 expressions and showed cytokine release, i.e., IL-2, IL-6, TNFα, and IFN-ɤ, at the concentration between 0.01 and 10 nM only when co-cultured with target cancer cells, however, those T cell activation and cytokine release were not observed under the conditions without target cancer cells, even at the concentration of 1 µM. In the presence of activated human PBMCs as effector cells, CB-1535 elicited 5T4 expressing target cell cytotoxicity with an EC50 of approximately 1-30 pM in a variety of cancer cells. The anti-tumor efficacy of CB-1535 has examined in immunocompromised mice bearing human lung mesothelioma (MSTO-211H) and alveolar epithelial adenocarcinoma (A549) xenografts (such as the 5T4 expressing target cell) and human PBMCs (as effector cells). Anti-tumor efficacy was observed with complete suppression of tumor growth at doses as low as 1 mg/kg in both xenograft models. No abnormalities related to CBA-1535 administration were observed regarding to body weight and clinical signs. Importantly, CBA-1535 showed a synergistic cytotoxicity against 5T4 expressing target cancer cells in vitro when combined with PD-1 antibody Pembrolizumab in a co-culture experiment. In conclusion, we demonstrated encouraging preclinical profile of CBA-1535 as a novel CD-3 based T-cell engager antibody. These results provide the strong rationale for further clinical evaluation of CBA-1535 in 5T4 positive tumors. CBA-1535 is now under the phase 1 trial in Japan (jRCT2031210708), with 2 parts, the monotherapy and the combination with Pembrolizumab. Citation Format: Fumitake Takizawa, Kotaro Yamamoto, Koji Nakamura, Ana R. Richard, Philip J. Cunnah, Nico Mertens, Pedro D. Pissarra, Yukihito Tsukumo. Preclinical characterization of CBA-1535, a novel bi-specific tribody, with two binding sites to 5T4 and one site to CD3ε [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 2937.

Abstract 6155: Lurbinectedin exhibits combinatorial activity with BCL2/BCL2L1 inhibitors in vitro and in vivo by modulation of MCL1 expression

Abstract Lurbinectedin (Zepzelca) is a marine-derived anti-tumor agent currently approved for metastatic small cell lung cancer (SCLC). Lurbinectedin has been previously shown to exert its anti-tumor effect by binding to the minor groove of DNA and inhibition of nucleotide excision repair. In addition, lurbinectedin inhibits transcription by inducing degradation of RNA polymerase II. While these activities hint towards a general mechanism, specific molecular targets for lurbinectedin remain obscure. We demonstrate that in vitro, Lurbinectedin specifically induced downregulation of MCL1 protein, a member of the BCL2 family of pro-survival proteins, in a dose- (0-30 nM) and time-dependent manner (0-24 h) by Western analysis. Expression of other pro-survival proteins BCL2 and BCL2L1 were not affected. The downregulation of MCL1 was consistent in all nine tumor cell lines tested regardless of indication. Increased activity or expression of MCL1 is a known compensatory mechanism to offset loss of BCL2 or BCL2L1. We therefore hypothesized that lurbinectedin induced MCL1 loss, along with inhibition of the pro-survival proteins BCL2 and BCL2L1 would lead to an enhancement in cell killing. Consistent with the dominant pro-survival role of BCL2 in SCLC, the combination of lurbinectedin with a specific BCL2 inhibitor (venetoclax) in vitro induced synergistic cytotoxicity (measured by Bliss Sum analysis) in a panel of small cell lung cancer lines. In solid tumor lines other than SCLC, the combination of lurbinectedin with a specific BCL2L1 inhibitor (A-1331852) was also synergistic. Correspondingly in vivo, enhancement in the efficacy of lurbinectedin was observed in combination with venetoclax in SCLC models, while in solid tumor models other than SCLC the combination with a specific BCL2L1 inhibitor enhanced efficacy. Collectively, these data identify MCL1 as a specific lurbinectedin target and suggest that combinations that target other pro-survival proteins may represent a viable strategy to enhance the anti-tumor activity of lurbinectedin. Citation Format: Kedar S. Vaidya, Aparna Gupta, Robert Hauptschein, Robin C. Humphreys. Lurbinectedin exhibits combinatorial activity with BCL2/BCL2L1 inhibitors in vitro and in vivo by modulation of MCL1 expression [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 6155.

Abstract 6104: Prognostic and therapeutic impact of a homologous recombination repair protein TONSL in cancer stem cells

Abstract Previously, we found that an important homologous recombination repair protein ‘Tonsoku Like, DNA Repair Protein’ (TONSL) might be required for glioblastoma cancer stem cell (CSC). In this study, we further investigated the role of TONSL in CSC from several cancer cell lines from different tissues. We also analyzed the clinical data available from the public databases to find the clinical impact of TONSL. The higher expression of TONSL is associated with the worse prognosis of lung and gastric cancer. Interestingly, in breast cancer, while its higher expression is also the negative prognostic marker of the luminal A and HER2 positive cancers, that is rather the positive prognostic marker for luminal B and basal cancers. TONSL expression is higher in all 4 subtypes of breast cancer, and ovarian cancer than the corresponding normal tissues. The knockdown of TONSL in an ovarian cancer cell line, OVCAR8, results in the increase of G2/M cells and apoptotic cells in the bulk cultured population while increase also the senescence cells only in the CSC population leading to the severe loss of CSC population. TONSL and the close binding partner in homologous recombination repair (HRR), MMS22L, both were essential for the CSC growth but the bulk cultured cells could survive without those two in a colon cancer cell line, HCT15 suggesting the importance of HRR in colon CSC. Indeed, gemcitabine treatment induced DNA breaks showed a synergistic cytotoxicity with those two HRR factors knockdown in the CSC only. These results suggest that the TONSL and MMS2L mediated HRR is essential for the CSC and while most of bulk cultured cancer cells circumvent the HRR deficit. Collectively, these data showed that the CSCs ultimately depend on the HRR process which requires TONSL and, therefore, CSC population may be more vulnerable to HRR targeting therapeutic approaches. Citation Format: Hani Lee, SeokGyeong Choi, Sojung Ha, Woo-Young Kim. Prognostic and therapeutic impact of a homologous recombination repair protein TONSL in cancer stem cells. [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 6104.

Abstract 6186: Patient-derived tumor organoids with p53 mutations, and not wild-type p53, demonstrate synergistic sensitivity to treatment with temozolomide in combination with talazoparib PARP inhibitor

Abstract Mutations in the TP53 gene can be found in more than 50% of tumors. We recently showed in 2D breast cancer cell lines that mutant p53 (mtp53) proteins tightly associate with replicating DNA, chromatin and Poly (ADP-ribose) polymerase (PARP) protein. In addition, missense mtp53 R273H causes an increase in the chromatin association of replication proteins including PARP, and mini-chromosome maintenance complex 2-7 (MCM2-7). The expression of mtp53 R273H enhances overall MCM2 levels, promotes cell proliferation, and improves the synergistic cytotoxicity of treatment with the alkylating agent temozolomide in combination with the PARP inhibitor (PARPi) talazoparib. Currently, PARPis are indicated for patients that present BRCA1/2 mutations, but there might be patients with other alterations associated with DNA damage repair that could benefit from PARPi treatment; this includes expression of mtp53. Patient-derived Tumor Organoids (PDTO) are 3D culture models that retain cell-cell and cell-matrix interactions and are shown to reproduce drug responses observed in patients. Here, we evaluated the sensitivity of wild-type BRCA1/2 breast and lung PDTO with either mtp53 or wild-type p53 (wtp53) to the combination of DNA damaging agent temozolomide plus the PARPi talazoparib. First, we tested the sensitivity of the organoids to talazoparib and temozolomide individually. To determine if there was synergy between the two treatments, the organoids were treated with an inhibitor matrix to evaluate multiple combinatorial concentrations. Three breast cancer and two lung cancer PDTO with mtp53 presented synergistic cytotoxicity of the combination treatment. Two breast organoid lines with wtp53 showed no synergistic interaction between the two drugs, and the same was observed in a wtp53 lung organoid line. We analyzed wtp53 and mtp53 PDTO cell extracts by western blot to assess the activation of downstream p53 effectors and DNA damage markers after treatment with temozolomide and/or talazoparib. Combination of talazoparib and temozolomide induced higher DNA double-strand breaks in mtp53 organoids as shown by increased gamma-H2AX expression. The results obtained demonstrated that in mtp53 PDTO synergism is achieved with combined talazoparib-temozolomide treatment supporting the idea that tumors expressing mtp53 may be potential candidates for this combination PARPi treatment. Citation Format: Florencia P. Madorsky Rowdo, Gu Xiao, Melissa B. Davis, Laura Martin, Olivier Elemento, Jill Bargonetti. Patient-derived tumor organoids with p53 mutations, and not wild-type p53, demonstrate synergistic sensitivity to treatment with temozolomide in combination with talazoparib PARP inhibitor. [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 6186.

Chloroquine synergizes doxorubicin efficacy in cervical cancer cells through flux impairment and down regulation of proteins involved in the fusion of autophagosomes to lysosomes

Drug repurposing holds abundant opportunity in the development of novel anticancer drugs. Chloroquine (CQ), a FDA approved anti-malarial drug, is demonstrated to enhance anticancer efficacy of standard anticancer drugs including doxorubicin (DOX) in several types of cancer cells. Here, we aimed to exploit the chemosensitizing effects of CQ against DOX in human cervical cancer (HeLa) cells that remains to be investigated yet. We show that a combination of DOX (40 nM) and CQ (40 μM) resulted in a synergistic cytotoxicity (combination index; CI < 1) in HeLa cells compared to the DOX or CQ alone. Synergistic effect of the combination (DOX + CQ) was associated with the impaired autophagic flux and enhanced apoptosis. Following treatment with the combination (DOX + CQ), the level of p62/SQSTM and LC-3II proteins was increased, while a decrease was noted in the expression of LAMP-2, Syntaxin17, Rab 5, and Rab 7 proteins that play critical roles in the fusion of autophagosomes to lysosomes. Autophagy inhibition by combination (DOX + CQ) enhanced the apoptotic cell death synergistically by increasing the cleavage of procaspase-3 and PARP1. Further, a prior incubation of HeLa cells with Z-VAD-FMK (a pan-caspase inhibitor) for 4 h, suppressed the combination (DOX + CQ)-induced cell death. Our data suggest that a combination of DOX + CQ had a better anti-cancer efficacy in HeLa cells than either of the drugs alone. Thus, CQ, as a repurposed drug, may hold the potential to synergize anticancer effects of DOX in cervical cancer cells.

Synthesis and Biological Activities of 11- and 12-Substituted Benzophenanthridinone Derivatives as DNA Topoisomerase IB and Tyrosyl-DNA Phosphodiesterase 1 Inhibitors.

Herein, a series of 11- or 12-substituted benzophenanthridinone derivatives was designed and synthesized for the discovery of dual topoisomerase IB (TOP1) and tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors. Enzyme-based assays indicated that two compounds 12 and 38 showed high TOP1 inhibitory potency (+++), and four compounds 35, 37, 39 and 43 showed good TDP1 inhibition with IC50 values ranging from 10 to 18 μM. 38 could induce cellular TOP1cc formation, resulting in the highest cytotoxicity against HCT-116 cells (0.25 μM). The most potent TDP1 inhibitor 43 (10 μM) could induce cellular TDP1cc formation and enhance topotecan-induced DNA damage and showed strong synergistic cytotoxicity with topotecan in both MCF-7 and MCF-7/TDP1 cells.

Co-delivery of paclitaxel and etoposide prodrug by human serum albumin and PLGA nanoparticles: synergistic cytotoxicity in brain tumour cells

The aims of this study were to develop co-delivery systems of paclitaxel (PTX) and etoposide prodrug (4’-O-benzyloxycarbonyl-etoposide, ETP-cbz) based on non-cross-linked human serum albumin (HSA) and poly(lactide-co-glycolide) nanoparticles and to evaluate the synergistic potential of these drugs in vitro. The nanoformulations were prepared by the high-pressure homogenisation technique and characterised using DLS, TEM, SEM, AFM, HPLC, CZE, in-vitro release, and cytotoxicity in human and murine glioma cells. All nanoparticles had 90-150 nm in size and negative ζ-potentials. The Neuro2A cells were the most sensitive to both HSA- and PLGA-based co-delivery systems (IC50 0.024 µM and 0.053 µM, respectively). The drugs’ synergistic effect (combination index < 0.9) was observed in the GL261 cells for both types of co-delivery formulations and in the Neuro2A cells for the HSA-based system. These nanodelivery systems may be useful to improve combination chemotherapy for brain tumour treatment. To our knowledge, this is the first report describing the non-cross-linked HSA-based co-delivery nanosuspension which was prepared using nab™ technology.

Individual and combined cytotoxicity effects of positively charged polystyrene nanoplastics and ionic surfactants on budding yeast Saccharomyces cerevisiae

It is of crucial importance to understand the impact of micro/nanoplastics contaminated with the other pollutants on microorganisms in the environment. In the present paper, we have investigated the individual and combined cytotoxicity effects of positively charged fluorescence-labeled polystyrene nanoparticles (pPS-NPs; 115 and 204 nm in diameter) and ionic surfactants [sodium dodecyl sulfate (SDS) and dodecyltrimethylammonium chloride (C12TAC)] on budding yeast Saccharomyces cerevisiae after a short-time exposure (0.5 h) in a 5-mM NaCl aqueous solution. In the absence of surfactants, the smaller 115-nm pPS-NPs were more cytotoxic than the larger 204-nm pPS-NPs. In the absence of pPS-NPs, the cationic surfactant of C12TAC was more cytotoxic than the anionic surfactant of SDS, though these two ionic surfactants have the hydrophobic alkyl chains of the same length and the oppositely charged headgroups in the same magnitude. The addition of SDS decreased the number of pPS-NPs adhered on a negatively charged yeast cell to reduce the cytotoxicity, whereby the combined toxicity effect was considered as counteracting action. Although some addition of C12TAC hardly influenced the number of pPS-NPs adhered on a yeast cell, the synergistic cytotoxicity action of the pPS-NPs and C12TAC was observed.

Auranofin Synergizes with the PARP Inhibitor Olaparib to Induce ROS-Mediated Cell Death in Mutant p53 Cancers

Auranofin (AF) is a potent, off-patent thioredoxin reductase (TrxR) inhibitor that efficiently targets cancer via reactive oxygen species (ROS)- and DNA damage-mediated cell death. The goal of this study is to enhance the efficacy of AF as a cancer treatment by combining it with the poly(ADP-ribose) polymerase-1 (PARP) inhibitor olaparib (referred to as 'aurola'). Firstly, we investigated whether mutant p53 can sensitize non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) cancer cells to AF and olaparib treatment in p53 knock-in and knock-out models with varying p53 protein expression levels. Secondly, we determined the therapeutic range for synergistic cytotoxicity between AF and olaparib and elucidated the underlying molecular cell death mechanisms. Lastly, we evaluated the effectiveness of the combination strategy in a murine 344SQ 3D spheroid and syngeneic in vivo lung cancer model. We demonstrated that high concentrations of AF and olaparib synergistically induced cytotoxicity in NSCLC and PDAC cell lines with low levels of mutant p53 protein that were initially more resistant to AF. The aurola combination also led to the highest accumulation of ROS, which resulted in ROS-dependent cytotoxicity of mutant p53 NSCLC cells through distinct types of cell death, including caspase-3/7-dependent apoptosis, inhibited by Z-VAD-FMK, and lipid peroxidation-dependent ferroptosis, inhibited by ferrostatin-1 and alpha-tocopherol. High concentrations of both compounds were also needed to obtain a synergistic cytotoxic effect in 3D spheroids of the murine lung adenocarcinoma cell line 344SQ, which was interestingly absent in 2D. This cell line was used in a syngeneic mouse model in which the oral administration of aurola significantly delayed the growth of mutant p53 344SQ tumors in 129S2/SvPasCrl mice, while either agent alone had no effect. In addition, RNA sequencing results revealed that AF- and aurola-treated 344SQ tumors were negatively enriched for immune-related gene sets, which is in accordance with AF's anti-inflammatory function as an anti-rheumatic drug. Only 344SQ tumors treated with aurola showed the downregulation of genes related to the cell cycle, potentially explaining the growth inhibitory effect of aurola since no apoptosis-related gene sets were enriched. Overall, this novel combination strategy of oxidative stress induction (AF) with PARP inhibition (olaparib) could be a promising treatment for mutant p53 cancers, although high concentrations of both compounds need to be reached to obtain a substantial cytotoxic effect.

Structure-activity relationship of hydroxycinnamate and carnosic acid derivatives for synergistic cytotoxicity to acute myeloid leukemia cells

Structure-activity relationship of hydroxycinnamate and carnosic acid derivatives for synergistic cytotoxicity to acute myeloid leukemia cells

Synergistic cytotoxicity effect of the combination of chitosan nanoencapsulated imatinib mesylate and quercetin in BCR-ABL positive K562 cells.

Intolerable side effects and resistance to chemotherapeutic drugs have encouraged scientists to develop new methods of drug combinations with fewer complications. This study aimed to investigate the synergistic effects of quercetin and imatinib encapsulated in chitosan nanoparticles on cytotoxicity, apoptosis, and cell growth of the K562 cell line. Imatinib and quercetin were encapsulated in chitosan nanoparticles and their physical properties were determined using standard methods and SEM microscope images. BCR-ABL positive K562 cells were cultured in a cell culture medium, cytotoxicity of drugs was determined by MTT assay and the effects of nano drugs on apoptosis in cells were investigated by Annexin V-FITC staining. The expression level of genes associated with apoptosis in cells was measured by real-time PCR. The IC50 for the combination of the nano drugs at 24 and 48 hr was 9.324 and 10.86 μg/ml, respectively. The data indicated that the encapsulated form of drugs induced apoptosis more effectively than the free form (P<0.05). Moreover, the synergistic effect of nano drugs in statistical analysis was proved (P=0.001). The combination of nano drugs resulted in the caspase 3, 8, and TP53 genes upregulation (P=0.001). The results of the present study showed that the encapsulated form of imatinib and quercetin nano drugs with chitosan has more cytotoxicity than the free form of the drugs. In addition, the combination of imatinib and quercetin as a nano-drug complex has a synergistic effect on the induction of apoptosis in imatinib-resistant K562 cells.

Antimicrobial and cytotoxic synergism of biocides and quorum-sensing inhibitors against uropathogenic Escherichiacoli

Uropathogenic Escherichia coli (UPEC) are a primary cause of catheter-associated urinary tract infections (CAUTIs), often forming mature recalcitrant biofilms on the catheter surface. Anti-infective catheter coatings containing single biocides have been developed but display limited antimicrobial activity due to the selection of biocide-resistant bacterial populations. Furthermore, biocides often display cytotoxicity at concentrations required to eradicate biofilms, limiting their antiseptic potential. Quorum-sensing inhibitors (QSIs) provide a novel anti-infective approach to disrupt biofilm formation on the catheter surface and help prevent CAUTIs. To evaluate the combinatorial impact of biocides and QSIs at bacteriostatic, bactericidal and biofilm eradication concentrations in parallel to assessing cytotoxicity in a bladder smooth muscle (BSM) cell line. Checkerboard assays were performed to determine fractional inhibitory, bactericidal, and biofilm eradication concentrations of test combinations in UPEC and combined cytotoxic effects in BSM cells. Synergistic antimicrobial activity was observed between polyhexamethylene biguanide, benzalkonium chloride or silver nitrate in combination with either cinnamaldehyde or furanone-C30 against UPEC biofilms. However, furanone-C30 was cytotoxic at concentrations below those required even for bacteriostatic activity. A dose-dependent cytotoxicity profile was observed for cinnamaldehyde when in combination with BAC, PHMB or silver nitrate. Both PHMB and silver nitrate displayed combined bacteriostatic and bactericidal activity below the half-maximum inhibitory concentration (IC50). Triclosan in combination with both QSIs displayed antagonistic activity in both UPEC and BSM cells. PHMB and silver in combination with cinnamaldehyde display synergistic antimicrobial activity in UPEC at non-cytotoxic concentrations, suggesting potential as anti-infective catheter-coating agents.

Novel construction of multifunctional photo-responsive and nucleic acid-triggered doxorubicin-releasing liposomes for cancer therapy

All-in-one nano theranostics integrating accurate diagnosis and combined therapy is promising for high-efficacy tumor treatment and receiving significant attention. In this study, we develop photo-controlled release liposomes with nucleic acid-triggered fluorescence and photoactivity for tumor imaging and synergistic antitumor therapy. Copper phthalocyanine as a photothermal agent is fused into lipid layers to prepare liposomes encapsulating cationic zinc phthalocyanine ZnPc(TAP)412+ and doxorubicin, followed by the modification of RGD peptide on the surface to obtain the final product RGD-CuPc:ZnPc(TAP)412+:DOX@LiPOs (RCZDL). RCZDL possesses favorable stability, significant photothermal effect, and photo-controlled release function through the characterization of physicochemical properties. It is shown that the fluorescence and ROS generation could be turned on by intracellular nucleic acid after illumination. RCZDL exhibits synergistic cytotoxicity, increased apoptosis, and significantly promoted cell uptake. Subcellular localization analysis indicates that ZnPc(TAP)412+ tends to be distributed in the mitochondria of HepG2 cells treated with RCZDL after exposure to light. The results of experiments in vivo on H22 tumor-bearing mice demonstrate that RCZDL had excellent tumor targeting, a prominent photothermal effect at the tumor sites, and synergistic antitumor efficiency. More importantly, little RCZDL has been found to be accumulated in the liver, and most were quickly metabolized by the liver. The results confirm that the proposed new intelligent liposomes provide a simple and cost-effective way for tumor imaging and combinatorial anticancer therapy.

Cytotoxic synergism of Clostridioides difficile toxin B with proinflammatory cytokines in subjects with inflammatory bowel diseases.

Clostridioides difficile (C. difficile) is progressively colonizing humans and animals living with humans. During this process, hypervirulent strains and mutated toxin A and B of C. difficile (TcdA and TcdB) are originating and developing. While in healthy subjects colonization by C. difficile becomes a risk after the use of antibiotics that alter the microbiome, other categories of people are more susceptible to infection and at risk of relapse, such as those with inflammatory bowel disease (IBD). Recent in vitro studies suggest that this increased susceptibility could be due to the strong cytotoxic synergism between TcdB and proinflammatory cytokines the tumor necrosis factor-alpha and interferon-gamma (CKs). Therefore, in subjects with IBD the presence of an inflammatory state in the colon could be the driver that increases the susceptibility to C. difficile infection and its progression and relapses. TcdB is internalized in the cell via three receptors: chondroitin sulphate proteoglycan 4; poliovirus receptor-like 3; and Wnt receptor frizzled family. Chondroitin sulphate proteoglycan 4 and Wnt receptor frizzled family are involved in cell death by apoptosis or necrosis depending on the concentration of TcdB and cell types, while poliovirus receptor-like 3 induces only necrosis. It is possible that cytokines could also induce a greater expression of receptors for TcdB that are more involved in necrosis than in apoptosis. Therefore, in subjects with IBD there are the conditions: (1) For greater susceptibility to C. difficile infection, such as the inflammatory state, and abnormalities of the microbiome and of the immune system; (2) for the enhancement of the cytotoxic activity of TcdB +Cks; and (3) for a greater expression of TcdB receptors stimulated by cytokines that induce cell death by necrosis rather than apoptosis. The only therapeutic approach currently possible in IBD patients is monitoring of C. difficile colonization for interventions aimed at reducing tumor necrosis factor-alpha and interferon-gamma levels when the infection begins. The future perspective is to generate bacteriophages against C. difficile for targeted therapy.

Smart Nanofiber Mesh with Locally Sustained Drug Release Enabled Synergistic Combination Therapy for Glioblastoma.

This study aims to propose a new treatment model for glioblastoma (GBM). The combination of chemotherapy, molecular targeted therapy and radiotherapy has been achieved in a highly simultaneous manner through the application of a safe, non-toxic, locally sustained drug-releasing composite Nanofiber mesh (NFM). The NFM consisted of biodegradable poly(ε-caprolactone) with temozolomide (TMZ) and 17-allylamino-17-demethoxygeldanamycin (17AAG), which was used in radiation treatment. TMZ and 17AAG combination showed a synergistic cytotoxicity effect in the T98G cell model. TMZ and 17AAG induced a radiation-sensitization effect, respectively. The NFM containing 17AAG or TMZ, known as 17AAG-NFM and TMZ-NFM, enabled cumulative drug release of 34.1% and 39.7% within 35 days. Moreover, 17AAG+TMZ-NFM containing both drugs revealed a synergistic effect in relation to the NFM of a single agent. When combined with radiation, 17AAG+TMZ-NFM induced in an extremely powerful cytotoxic effect. These results confirmed the application of NFM can simultaneously allow multiple treatments to T98G cells. Each modality achieved a significant synergistic effect with the other, leading to a cascading amplification of the therapeutic effect. Due to the superior advantage of sustained drug release over a long period of time, NFM has the promise of clinically addressing the challenge of high recurrence of GBM post-operatively.

Discovery of β-cyclocitral-derived mono-carbonyl curcumin analogs as anti-hepatocellular carcinoma agents via suppression of MAPK signaling pathway

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with a high recurrence and mortality rate. In this study, a series of β-cyclocitral-derived mono-carbonyl curcumin analogs were synthesized and their anticancer properties were evaluated. Among the series, A19 exhibited the strongest cytotoxic activity by inhibiting cell viability and colony formation, inducing cell cycle G2/M phase arrest and cell apoptosis of HCC HepG2 and Huh-7 cells, while having almost no cytotoxicity on normal liver MIHA cells. Mechanistically, our results demonstrated that A19 triggered intense DNA damage via suppression of the ERK/JNK/p38 MAPK signaling pathway. Additionally, a combination of A19 with sorafenib significantly induced synergistic cytotoxicity in HCC cells. Overall, our results indicate the potential of A19 as a novel chemotherapeutic drug administered either separately or in combined therapy for HCC treatment.