Low Concentrations Of Cisplatin Research Articles

Combination Treatment of Withalongolide a Triacetate with Cisplatin Induces Apoptosis by Targeting Translational Initiation, Migration, and Epithelial to Mesenchymal Transition in Head and Neck Squamous Cell Carcinoma.

Treatment regimens for head and neck squamous cell carcinoma (HNSCC) typically include cisplatin and radiotherapy and are limited by toxicities. We have identified naturally derived withalongolide A triacetate (WGA-TA) from Physalis longifolia as a lead compound for targeting HNSCC. We hypothesized that combining WGA-TA with cisplatin may allow for lower, less toxic cisplatin doses. HNSCC cell lines were treated with WGA-TA and cisplatin. After treatment with the drugs, the cell viability was determined by MTS assay. The combination index was calculated using CompuSyn. The expression of proteins involved in the targeting of translational initiation complex, epithelial to mesenchymal transition (EMT), and apoptosis were measured by western blot. Invasion and migration were measured using the Boyden-chamber assay. Treatment of MDA-1986 and UMSCC-22B cell lines with either WGA-TA or cisplatin alone for 72 h resulted in a dose dependent decrease in cell viability. Cisplatin in combination with WGA-TA resulted in significant synergistic cell death starting from 1.25 μM cisplatin. Combination treatment with WGA-TA resulted in lower cisplatin dosing while maintaining the downregulation of translational initiation complex proteins, the induction of apoptosis, and the blockade of migration, invasion, and EMT transition. These results suggest that combining a low concentration of cisplatin with WGA-TA may provide a safer, more effective therapeutic option for HNSCC that warrants translational validation.

Extrinsic apoptosis and senescence involved in growth kinetics of seminoma to cisplatin.

Seminoma is the most common type of testicular germ cell tumour and is highly sensitive to cisplatin therapy, which has not been fully elucidated. In this study, we comprehensively monitored dynamic changes of TCam-2 cells after cisplatin treatment. At an early stage, we found that both low and high concentrations of cisplatin induced the S-phase arrest in TCam-2 cells. By contrast, the G0G1 arrest was caused by cisplatin in teratoma NTERA-2 cells. Afterwards, high concentrations of cisplatin promoted the extrinsic apoptosis and high expressions of related genes (Fas/FasL-caspase-8/-3) in TCam-2 cells. However, when decreasing the cisplatin, the apoptotic cells were significantly reduced, and accompanied by cells showing senescence-like morphology, positive SA-β-gal staining and up-regulation of senescence-related genes (p53, p21 and p16). Furthermore, the cell cycle analysis revealed that most of the senescent TCam-2 cells were irreversibly arrested in the G2M phase. G2M arrest was also observed in NTERA-2 cells treated with a low concentration of cisplatin, while no senescence-related phenotype was discovered. In addition, we detected the γ-H2AX, a DNA damage marker protein, and reactive oxygen species (ROS) and found both of which were elevated with the increase of cisplatin in TCam-2 cells. In conclusion, the extrinsic apoptosis and senescence are involved in the growth kinetics of TCam-2 cells to cisplatin, which provide some implications for studies on cisplatin and seminoma.

Dynamic Phenotypic Switching and Group Behavior Help Non-Small Cell Lung Cancer Cells Evade Chemotherapy.

Drug resistance, a major challenge in cancer therapy, is typically attributed to mutations and genetic heterogeneity. Emerging evidence suggests that dynamic cellular interactions and group behavior also contribute to drug resistance. However, the underlying mechanisms remain poorly understood. Here, we present a new mathematical approach with game theoretical underpinnings that we developed to model real-time growth data of non-small cell lung cancer (NSCLC) cells and discern patterns in response to treatment with cisplatin. We show that the cisplatin-sensitive and cisplatin-tolerant NSCLC cells, when co-cultured in the absence or presence of the drug, display dynamic group behavior strategies. Tolerant cells exhibit a ‘persister-like’ behavior and are attenuated by sensitive cells; they also appear to ‘educate’ sensitive cells to evade chemotherapy. Further, tolerant cells can switch phenotypes to become sensitive, especially at low cisplatin concentrations. Finally, switching treatment from continuous to an intermittent regimen can attenuate the emergence of tolerant cells, suggesting that intermittent chemotherapy may improve outcomes in lung cancer.

The proteolytic landscape of cells exposed to non-lethal stresses is shaped by executioner caspases

Cells are in constant adaptation to environmental changes to insure their proper functioning. When exposed to stresses, cells activate specific pathways to elicit adaptive modifications. Those changes can be mediated by selective modulation of gene and protein expression as well as by post-translational modifications, such as phosphorylation and proteolytic processing. Protein cleavage, as a controlled and limited post-translational modification, is involved in diverse physiological processes such as the maintenance of protein homeostasis, activation of repair pathways, apoptosis and the regulation of proliferation. Here we assessed by quantitative proteomics the proteolytic landscape in two cell lines subjected to low cisplatin concentrations used as a mild non-lethal stress paradigm. This landscape was compared to the one obtained in the same cells stimulated with cisplatin concentrations inducing apoptosis. These analyses were performed in wild-type cells and in cells lacking the two main executioner caspases: caspase-3 and caspase-7. Ninety-two proteins were found to be cleaved at one or a few sites (discrete cleavage) in low stress conditions compared to four hundred and fifty-three in apoptotic cells. Many of the cleaved proteins in stressed cells were also found to be cleaved in apoptotic conditions. As expected, ~90% of the cleavage events were dependent on caspase-3/caspase-7 in apoptotic cells. Strikingly, upon exposure to non-lethal stresses, no discrete cleavage was detected in cells lacking caspase-3 and caspase-7. This indicates that the proteolytic landscape in stressed viable cells fully depends on the activity of executioner caspases. These results suggest that the so-called executioner caspases fulfill important stress adaptive responses distinct from their role in apoptosis. Mass spectrometry data are available via ProteomeXchange with identifier PXD023488.

Inhibition of the Notch signaling pathway attenuates progression of cell motility, metastasis, and epithelial-to-mesenchymal transition-like phenomena induced by low concentrations of cisplatin in osteosarcoma

Although advances in osteosarcoma treatment have been made in recent decades, the survival rate for patients suffering from metastatic disease, especially lung metastasis, remains disappointing. Previous studies have confirmed that epithelial-to-mesenchymal transition (EMT) is associated with tumor metastasis, and several studies have suggested that osteosarcoma cells also exhibit EMT-like characteristics. In addition, Notch signaling is known to be related to the development and progression of human malignancies, including osteosarcoma. However, whether chemotherapy affects the EMT-like events and whether these events are medicated by Notch signaling remain to be elucidated. To address these issues, in the current work, osteosarcoma 143B cells were exposed to sublethal concentrations of the first-line chemotherapeutic agent cisplatin (DDP), which promoted cell migration, in vitro invasion, and in vivo lung metastasis. Furthermore, low concentrations of DDP upregulated mesenchymal phenotype-related genes and proteins and promoted EMT-like properties in osteosarcoma cells. In addition, low concentrations of DDP could activate the Notch receptor and its target genes. Finally, combined treatment of DDP with the Notch signaling pathway inhibitor DAPT, which can effectively downregulate mesenchymal phenotype-related genes and proteins, inhibited cell migration and invasion in vitro, and it decreased pulmonary metastatic nodules in vivo. The results of the current study supported the idea that low concentrations of DDP could induce EMT-like characteristics in osteosarcoma cells and could promote cell mobility in vitro, as well as pulmonary metastasis in vivo. Importantly, however, these biological processes are mediated by the Notch signaling pathway. Blocking the Notch signaling pathway can effectively attenuate the osteosarcoma EMT-like phenotype and its associated migration, invasion, and metastasis.

G protein-coupled receptor kinase 2 modifies the cellular reaction to cisplatin through interactions with NADPH oxidase 4.

G protein-coupled receptor kinases (GRKs), in addition to their role in modulating signal transduction mechanisms associated with activated G protein-coupled receptors (GPCRs), can also interact with many non-GPCR proteins to mediate cellular responses to chemotherapeutics. The rationale for this study is based on the presumption that GRK2 modulates the responses of cancer cells to the chemotherapeutic cisplatin. In this report, we show that GRK2 modulates the responses of cancer cells to cisplatin. Cervical cancer HeLa cells stably transfected with GRK2 shRNA, to decrease GRK2 protein expression, show increased sensitivity to cisplatin. Of interest, these cells also show increased accumulation of NADPH, associating with decreased NADP buildup, at low concentrations of cisplatin tested. These changes in NADPH and NADP levels are also observed in the breast cancer MDA MB 231 cells, which has lower endogenous GRK2 protein expression levels, but not BT549, a breast cancer cell line with higher GRK2 protein expression. This effect of NADPH accumulation may be associated with a decrease in NADPH oxidase 4 (NOX4) protein expression, which is found to correlate with GRK2 protein expression in cancer cells-a relationship which mimics that observed in cardiomyocytes. Furthermore, like in cardiomyocytes, GRK2 and NOX4 interact to form complexes in cancer cells. Collectively, these results suggest that GRK2 interacts with NOX4 to modify cisplatin sensitivity in cancer cells and may also factor into the success of cisplatin-based regimens.

A Preliminary Study: The Selective Effect of Bee Venom on Inhibition of Cell Migration in Metastatic Breast Cancer Cells Over Normal Cells in Comparison with Cisplatin

Bee venom is considered as an effective alternative medication in the treatment of some diseases, such cancer, but it has not been applied to patients routinely. Bee venom has in vitro selective cytotoxic effect on some cancer cells examined but the mechanism of action has not been fully elucidated. Cancer is characterized by not only uncontrolled and increased cell proliferation but also by invasive and metastatic properties of proliferating cells. Wound healing assay is used for revealing the migration rate of cells in vitro. The effect of bee venom on healing diabetes-induced wound has been shown before, but to the best of knowledge there is no study showing migration velocity of metastatic breast cancer cells after bee venom treatment. This study aimed (i) to reveal the metastatic behaviour of metastatic breast cancer cells after bee venom compared to cisplatin, and (ii) also to conclude the differences between normal and cancer cells in terms of migration after bee venom and cisplatin. Findings showed that bee venom was more effective on selective inhibition of cancer cell migration, but cisplatin-mediated inhibitory effect on metastasis could not be detected as even low concentrations of cisplatin induced high rate of cell death. The preliminary findings need to be expanded to cover more cell lines and the mechanism for this effect should be disclosed by detailed molecular methodologies for the future as well.

Dynamic Phenotypic Switching and Group Behavior Help Cancer Cells Evade Chemotherapy

Drug resistance, which is a major challenge in cancer therapy, is typically attributed to mutations and genetic heterogeneity. However, emerging evidence suggests that dynamic cellular interactions and group behavior also contribute to drug resistance, although, the underlying mechanisms remain poorly understood. Here, we developed a new mathematical approach with game theoretical underpinnings to model extensive real time growth data to discern patterns in group behavior. We show that the cisplatin-sensitive and cisplatin-tolerant lung cancer cells when co-cultured in the absence or presence of cisplatin, display dynamic group behavior strategies. Tolerant cells exhibit a persister-like behaviour and are attenuated by sensitive cells; they can also ‘educate’ sensitive cells to evade chemotherapy. Further, tolerant cells can switch phenotypes to become sensitive, especially at low cisplatin concentrations. Finally, switching treatment from continuous to an intermittent regimen, can attenuate the emergence of tolerant cells suggesting that intermittent chemotherapy may improve outcomes in lung cancer.

Synthesis, Anticancer Evaluation and Synergistic Effects with cisplatin of Novel Palladium Complexes: DNA, BSA Interactions and Molecular Docking Study.

In order to discover new agents for chemotherapy with improved properties compared to the existing agents and bearing in mind the fact that some Pd complexes possess better antitumor activity and exhibit less kidney toxicity compared to cisplatin, a series of novel square-planar palladium(II) complexes [Pd (L)2] (3a-f) with O,O bidentate ligands [L = ethyl 2- hydroxy-alkyl(aryl)-4-oxo-2-butenoate] were synthesized. All complexes were characterized by spectral (UV-Vis, IR, NMR, ESI-MS) and X-ray analysis and examined for their cytotoxic effect on human cancer cell lines HeLa and MDA-MB 231 and normal fibroblasts (MRC-5). Fluorescence spectroscopic method was used for investigations of the interactions between CT-DNA or bovine serum albumin (BSA) and complex 3c. Viscosity measurements and molecular docking study were performed to confirm the mode of interactions between DNA and BSA and complex 3c. Complexes that showed the best results, 3c, 3d, and 3e, were placed under further investigations. Selected complexes induced apoptosis and cell cycle arrest in HeLa and MDA-MB 231 cells. Low concentrations of 3c and 3e showed strong to moderate synergism with low concentrations of cisplatin. The interaction of 3d with cisplatin was antagonistic in all used concentrations, but low IC50 value indicates its usefulness as a single cytotoxic agent. It was also noted that the change of viscosity is more pronounced in DNA solution after addition of complex 3c. Obtained results indicate that the novel palladium(II) complexes have the potential to become candidates for treatment in anticancer therapy.

ATR kinase inhibition sensitizes quiescent human cells to the lethal effects of cisplatin but increases mutagenesis

The ATR protein kinase is known to protect cells from DNA damage induced during the replicative phase of the cell cycle. Small molecule ATR kinase inhibitors have therefore been developed to improve the effectiveness of DNA damage-based chemotherapy regimens aimed at killing rapidly proliferating tumor cells. However, whether ATR functions in a similar manner in non-replicating cells has not been examined and is important considering the fact that most cells in the body, including cancer stem cells in solid tumors, normally reside in either a quiescent or differentiated non-replicating state. Using cultured human cell lines maintained in a quiescent or slowly growing state in vitro, ATR was found to be activated following treatment with the common anti-cancer drug cisplatin in a manner dependent on the nucleotide excision repair (NER) system. Moreover, treatment with the ATR kinase inhibitors VE-821 and AZD6738 enhanced quiescent cell killing and apoptotic signaling induced by cisplatin. However, ATR kinase inhibition in quiescent cells treated with a low concentration of cisplatin also elevated the level of mutagenesis at the hypoxanthine phosphoribosyltransferase locus and resulted in increased levels of PCNA mono-ubiquitination. These results suggest that the excision gaps generated by NER may require a greater utilization of potentially mutagenic translesion synthesis polymerases in the absence of ATR kinase function. Thus, though ATR kinase inhibitors can aid in the killing of cisplatin-treated quiescent cells, such treatments may also result in a greater reliance on alternative mutagenic DNA polymerases to complete the repair of cisplatin-DNA adducts.

Profiling of apoptosis- and autophagy-associated molecules in human lung cancer A549 cells in response to cisplatin treatment using stable isotope labeling with amino acids in cell culture.

Cis‑diammine‑dichloro‑platinum II‑based adjuvant chemotherapy provides an alternative therapy to improve the survival of patients with lung tumors, especially those with non‑small cell lung cancer (NSCLC). However, drug resistance is a large clinical problem and its underlying mechanism remains unclear. In the present study, NSCLC A549 cells were treated with a low concentration of cisplatin in order to observe and determine the development of chemoresistance, via growth curves, colony forming assays and apoptosis assays. Then the induction of autophagy was examined in the cisplatin‑treated A549 cells with a fluorescence reporter. Profiled proteins in the cisplatin‑treated A549 cells were also assessed using the stable isotope labeling by amino acids in cell culture (SILAC) method, and then the differentially expressed molecules were verified. The results demonstrated that A549 cells became less sensitive to cisplatin [resistant A549 cells (A549R)] following 20passages in the medium containing a low concentration of cisplatin, with less apoptotic cells post‑cisplatin treatment. A549R cells grew more efficiently in the cisplatin medium, with more colony formation and more cells migrating across the baseline. In addition, NSCLC results demonstrated that more autophagy‑related proteins (ATGs) were expressed in the A549R cells. Furthermore, the western blotting results confirmed this upregulation of ATGs in A549R cells. In addition, two repeated SILAC screening experiments recognized 15proteins [glucose‑regulated protein, 78kDa (GRP78), heat shock protein71, pre‑mRNA processing factor19, polypyrimidine tract binding protein1, translationally controlled tumor protein, CathepsinD, Cytochromec, thioredoxin domain containing5, MutS homolog (MSH)6, AnnexinA2 (ANXA2), BRCA2 and Cyclin dependent kinase inhibitor1A interacting protein, MSH2, protein phosphatase2A 55kDa regulatory subunitBα, Rho glyceraldehyde‑3‑phosphate‑dissociation inhibitor1 and ANXA4] that were upregulated by >1.5‑fold in heavy(H)‑ and light(L)‑labeled A549R cells. In addition, 16 and 14proteins were downregulated by >1.5‑fold in the H‑ and L‑labeled A549R cells, respectively. The majority of the downregulated proteins were associated with apoptosis. In conclusion, the present study isolated a cisplatin‑resistant human lung cancer A549 cell clone, with reduced apoptosis and high levels of autophagy, in response to cisplatin treatment. In cisplatin‑resistant A549R cells, SILAC proteomics recognized the high expression of GRP78 and other proteins that are associated with anti‑apoptosis and/or autophagy promotion.

MicroRNA‑29a enhances cisplatin sensitivity in non‑small cell lung cancer through the regulation of REV3L.

Cisplatin-based chemotherapy may greatly enhance patient prognosis; however, chemotherapy resistance remains an obstacle to curing patients with non-small cell lung cancer (NSCLC). The aim of the present study was to explore the microRNAs (miRs) that could regulate cisplatin sensitivity and provide a potential treatment method for cisplatin resistance in clinical. Results from the present study revealed that miR-29a overexpression enhanced and miR-29a inhibition reduced the sensitivity of two NSCLC cell lines, A549 and H1650, to cisplatin treatment. In addition, reduced miR-29a expression levels were observed in cisplatin-resistant A549 cells (A549rCDDP), and increased expression of miR-29a augmented cisplatin-induced inhibition of proliferation and apoptosis in A549rCDDP cells. These data indicated that miR-29a expression may be involved in the development of cisplatin resistance. miR-29a was revealed to negatively regulate REV3-like DNA-directed polymerase ζ catalytic subunit (REV3L) expression in both A549 and H1650 cells; elevated expression of REV3L in A549rCDDP cells was also detected. REV3L encodes the catalytic subunit of DNA polymerase ζ and was hypothesized, based on results from the online tool TargetScan 7.1, to be a target gene of miR-29a; this was confirmed with a dual luciferase assay. Cells treated with a very low concentration of cisplatin exhibited a significant reduction in proliferation and cell cycle arrest at the G2/M phase in REV3L-knockdown as well as in miR-29a-upregulated A549 cells. Notably, reduced miR-29a expression and an increase in REV3L mRNA expression were observed in tumor tissues from patients with NSCLC. Additionally, a negative correlation between miR-29a and REV3L mRNA expression levels in tumor tissues from patients with NSCLC was observed; low expression of miR-29a and high expression of REV3L were closely associated with an advanced tumor-node-metastasis classification. The results of the present study suggested a pivotal role of miR-29a in mediating NSCLC cell sensitivity towards cisplatin through the regulation of REV3L.

Long non-coding RNA TUG1 sponges miR-197 to enhance cisplatin sensitivity in triple negative breast cancer

Breast cancer is the leading cause of women death worldwide. Several long non-coding RNAs (lncRNAs) have been identified as oncogenes or tumor suppressors during the progression of cancers. However, the role of taurine upregulated gene (TUG1) in mediating the chemotherapy sensitivity of triple negative breast cancer (TNBC) has not been studied yet. In TNBC patients, we observed a significant decrease of TUG1 in tumor tissues compared to the normal tissues. Similarly, TUG1 expression was significantly decreased in TNBC cell lines compared with normal breast epithelial cell line and cell lines of other subtypes of breast cancer. In MDA-MB-231 and BT549, cisplatin induced cell growth arrest was remarkably augmented by overexpression of TUG1 and was significantly reduced by TUG1 silencing. Moreover, very low concentration of cisplatin caused cell proliferation inhibition in TUG1-overexpressed-TNBC cells. In addition, we found that TUG1 negatively regulated miR-197 expression in the tested TNBC cell lines. Sponging of TUG1 to miR-197 was proved by a dual luciferase reporter assay. We further predicted and validated that nemo-like kinase (NLK), which was positively controlled by TUG1, was a target gene of miR-197. Via regulation of miR-197/NLK, TUG1 inactivated WNT signaling pathway and thus increasing chemotherapy sensitivity of TNBC cells. Analysis of TCGA database showed that higher expression of TUG1 was associated with better prognosis in breast cancer patients. Our current study drew a preliminary conclusion that TUG1 was involved in chemotherapy sensitivity in TNBC cells.

Synthesis of cell penetrating peptide decorated magnetic nanoparticles loading cisplatin for nasopharyngeal cancer therapy

Objective:To synthesize cisplatin loaded and cell penetrating peptide TAT decorated magnetic nanoparticles and to observe the inhibiting effect in vitro on nasopharyngeal cancer therapy.Method：The aldehyde sodium alginate coated magnetic nanoparticles (ASA-MNPs) was prepared as the drug delivery system, which was covalently attached by PEGylation TAT (TAT-ASA-MNPs) via condensation of aldehyde with amino group and then coordinated with cisplatin (TAT-ASA-MNPs@CDDP). The complex was characterized by H-NMR and FT-IR. The cell penetrating ability and biocompatibility were observed by means of fluorescent tags. The inhibited effect on nasopharyngeal cancer CNE-2 cells was measured by cellular toxicity research and flow cytometry.Result：The H NMR and FT-IR of TAT-ASA-MNPs exhibited the characteristic peaks of TAT, PEG as well as ASA. The dynamic light scattering showed the hydrodynamic diameter of the complex was（145.9±1.5）nm. Zeta potential was（-21.66±1.24）mV and the drug loading rate was（25.03±3.05）%. Fluorescent labeling assay revealed that FITC marked TATASAMNPs was quickly taken up by CNE-2 cells. Cytotoxicity experiment on 293T cells displayed high survival rate (>70%) after cultured for 72h. Negative hemagglutination reflected decent biocompatibility. In vitro cytotoxicity test and cell apoptosis assay exhibited obvious inhibition on CNE-2 cell with TATASAMNPs@CDDP at low concentration of cisplatin compared to ASA-MNPs@CDDP (P<0.05).Conclusion：TAT-ASA-MNPs showed decent biocompatibility while distinctly inhibit CNE-2 cells in vitro study.

In Vitro Genotoxic Effects of Zerumbone and Cisplatin Combination in CHO Cell Lines

Objective: Several natural products are being increasingly used in the treatment of cancer to minimize the adverse side effects of cancer chemotherapy. Zerumbone (ZER), the sesquiterpene derived from Zingiber zerumbet Smith, has been reported to have an in vitro anticancer effects against various human tumour cells as well as in vivo against a number of induced malignancies in mice. Previously we have reported the genotoxic effects of ZER in vitro against CHO cell lines. Material and Method: The aim of this study was to investigate the genotoxic effects of the combination of ZER along with cisplatin in CHO cells. Two cytogenetic endpoints were used, namely Chromosomal Aberrations assay (CA) and Micronucleus test (MN). Both cytogenetic endpoints were performed without any metabolic activation. Result: ZER treated cultures showed the significant increase in the frequency of the chromosome aberrations and MN induction. In CA assay, marked changes have been observed after co-treatment of CHO cell lines with different concentration of ZER along with 5 µM Cisplatin when compared to ZER treatment alone, suggesting a possible synergistic genetoxic effects. Whereas, treatment of CHO cell lines with different concentrations of ZER along with 2.5 µM Cisplatin was found to reduce chromosomal aberrations, suggesting an antagonistic genotoxic effect. On the other hand, in MN induction test, co-treatment of CHO cell lines with both 2.5 µM and 5 µM Cisplatin and different concentration of ZER found to reduce the genotoxic effects compared to the 2.5 µM and 5 µM Cisplatin alone suggesting an antagonistic genotoxic effect.Conclusion: The genotoxic effects of combined low concentrations of Cisplatin with different concentrations of ZER could have an antagonist genotoxic potential in vitro in CHO cell lines.

Ovarian cancer spheroid shrinkage following continuous exposure to cisplatin is a function of spheroid diameter

ObjectiveMost ovarian cancer patients present with advanced-stage disease, disseminated in the peritoneal cavity. Standard treatment involves surgical resection of all visible tumor, followed by delivery of systemic therapy. Patients with advanced-stage disease may be candidates for intraperitoneal (IP) chemotherapy following surgical debulking. Recent clinical trials have created controversy regarding the benefits of this approach. Previous clinical trials report that patients with microscopic residual disease respond best to IP therapy. The goal of this study was to determine the relationship between tumor size and the efficacy of continuous chemotherapy. MethodsSmall and large ovarian cancer spheroids (derived from UCI101 and A2780 cell lines) were exposed to short-term high (modeling an IP injection, “IP”) or prolonged, low cisplatin concentrations (modeling an implanted device, “device”), which have been previously shown to be less toxic. Spheroid diameter was measured at various time points via image analysis and used to quantify tumor shrinkage over the course of treatment. ResultsWe show that “IP” doses more effectively shrink large spheroids when the same cumulative dose is administered with both treatments, but that both regimens similarly treat small spheroids. We also demonstrate that higher cumulative “device” doses are most effective at shrinking large spheroids. ConclusionsThese results support the hypothesis that intratumoral drug distribution following IP treatment is diffusion-controlled. An implanted device that continuously delivers low doses of IP chemotherapy would, therefore, be maximally effective against microscopic tumors.

Abstract B26: A novel naturally-derived withalonglide synergizes with cisplatin to block self-renewal, migration, and EMT transition to enhance apoptosis via targeting of translational initiation

Abstract Introduction: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world with greater than 300,000 fatalities annually. Despite advances in surgery, chemotherapy and radiotherapy, the five-year survival rate of HNSCC has not changed much over the last four decades due to recurrence and metastasis. Current first-line standard treatment for advanced HNSCC patients is chemotherapy with a platinum agent (cisplatin) and radiotherapy, which carry problems with resistance and toxicity, creating a critical need for novel therapeutics that are not toxic, more efficacious or can mitigate cisplatin limitations. Through structure activity relationships we have identified and characterized a novel, naturally derived withalongolide A-triacetate (WGA-TA) from Physalis longifolia as a lead compound for targeting several cancers including HNSCC. WGA-TA selectively targets several key pathways including PI3K/mToR through modulation of HSP90-cdc37 chaperon function and has a high therapeutic index. Therefore, we hypothesize that combining WGA-TA with cisplatin can be synergistic and lower toxicity in combination. Methods: Validated HNSCC cell lines MDA-1986, UMSCC-22B and UMSCC-12 were treated with serial dilutions of WGA-TA and cisplatin starting from 10 nM to 100 μM and the viability of the cells were determined by MTS assay. The IC50 values for single drug treatment was determined using GraphPad Prism. Based on the IC50 values the single drug treatment cells were treated with varying combinations of cisplatin and WGA-TA that are above their IC50 values and the combination index (CI) was calculated using CompuSyn. CI&amp;lt;1 was considered synergistic. Targeting of translational initiation complex proteins an as well as the EMT transition was measured after treating MDA-1986 and UMSCC 22B cells with the combinations that were synergistic. Induction of apoptosis was measured by western blot analysis for the cleavage of PARP and further validated by Flow cytometry. Self-renewal was assessed by sphere formation assay. Boyden-chamber assay was used to measure invasion and migration. Results: When HNSCC cells were treated with varying concentrations of either WGA-TA alone or cisplatin alone for 72 h, a dose dependent decrease in viability was observed. The IC50 value for WGA-TA treatment was 1.7μM, 0.611μM and 0.75μM and that for cisplatin treatment was 6.639 μM, 4.141 μM and 5 μM for UMSCC-12, MDA1986 and UMSCC-22B respectively. When cells were treated with varying combinations of cisplatin and WGA-TA for 72 h or 24 h, we observed CI value of &amp;lt;1 when several different concentrations of WGA-TA are combined with 1.25 and 2.5 μM cisplatin for both MDA-1986 and UMSCC-22B indicating that combining cisplatin with WGA-TA has synergistic effect (p&amp;lt;0.01). To further evaluate whether WGA-TA in combination with cisplatin could target translational initiation complex proteins, we have treated UMSCC-22B and MDA-1986 with either 1.25 μM cisplatin or 2.5 μM cisplatin along with varying concentrations of WGA-TA for 24 h and evaluated modulation of translational complex proteins by western blot analysis. Our results indicated down regulation of p-e1F4B by 50-90%, pe1F4G by 80-90% and p4EBP by 45-70% for combination treatments with no changes in e1F4B, e1F4G, 4-EPB1, e1F4E, pe1F4E, e1F4A and e1F4A1. Consistent with down regulation of translational initiation, PARP cleavage as well as decrease in the levels of vimentin and increase in the expression of e-cadherin was also observed by immuno blot analysis indicating induction of apoptosis and blocking of EMT transition. Finally, we demonstrate that cancer stem cell functions such as self-renewal, migration and invasion was also blocked significantly using the combination therapy. Conclusion: WGA-TA when combined with cisplatin synergizes to effectly target translational initiation complex proteins. Enhanced apoptosis observed while combining very low concentration of cisplatin with WGA-TA indicates that the combination treatment is a much safer and effective option for targeting HNSCC that mitigates the dose and toxicity associated with standard cisplatin treatment. Further in vivo validation studies are warranted to define this synergistic effect for rapid clinical translation. Citation Format: Chitra Subramanian, Robin Pierce, Michaela Clague, Barbara N. Timmermann, Mark S. Cohen. A novel naturally-derived withalonglide synergizes with cisplatin to block self-renewal, migration, and EMT transition to enhance apoptosis via targeting of translational initiation. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B26.

Inhibition of endoplasmic reticulum (ER) stress sensors sensitizes cancer stem-like cells to ER stress-mediated apoptosis.

Although cancer stem cells (CSC) have been implicated in the development of resistance to anti-cancer therapy including chemotherapy, the mechanisms underlying chemo-resistance by CSC have not yet been elucidated. We herein isolated sphere-forming (cancer stem-like) cells from the cervical cancer cell line, SiHa, and examined the unfolded protein reaction (UPR) to chemotherapeutic-induced endoplasmic reticulum (ER) stress. We revealed that tunicamycin-induced ER stress-mediated apoptosis occurred in monolayer, but not sphere-forming cells. Biochemical assays demonstrated that sphere-forming cells were shifted to pro-survival signaling through the inactivation of IRE1 (XBP-1 splicing) and activation of PERK (elF2α phosphorylation) branches under tunicamycin-induced ER stress conditions. The proportion of apoptotic cells among sphere-forming cells was markedly increased by the tunicamycin+PERK inhibitor (PERKi) treatment, indicating that PERKi sensitized sphere-forming cells to tunicamycin-induced apoptosis. Cisplatin is also known to induce ER stress-mediated apoptosis. A low concentration of cisplatin failed to shift sphere-forming cells to apoptosis, although IRE1 branch, but not PERK, was activated. ER stress-mediated apoptosis occurred in sphere-forming cells by the cisplatin+IRE1α inhibitor (IRE1i) treatment. IRE1i, synergistic with cisplatin, up-regulated elF2α phosphorylation, and this was followed by the induction of CHOP in sphere-forming cells. The results of the present study demonstrated that the inhibition of ER stress sensors, combined with ER stress-inducible chemotherapy, shifted cancer stem-like cells to ER stress-mediated apoptosis.

Using Single‐Cell Amperometry To Reveal How Cisplatin Treatment Modulates the Release of Catecholamine Transmitters during Exocytosis

AbstractThe pretreatment of cultured pheochromocytoma (PC12) cells with cis‐diamminedichloroplatinum (cisplatin), an anti‐cancer drug, influences the exocytotic ability of the cells in a dose‐dependent manner. Low concentrations of cisplatin stimulate catecholamine release whereas high concentrations inhibit it. Single‐cell amperometry reflects that 2 μm cisplatin treatment increases the frequency of exocytotic events and reduces their duration, whereas 100 μm cisplatin treatment decreases the frequency of exocytotic events and increases their duration. Furthermore, the stability of the initial fusion pore that is formed in the lipid membrane during exocytosis is also regulated differentially by different cisplatin concentrations. This study thus suggests that cisplatin influences exocytosis by multiple mechanisms.

Using Single‐Cell Amperometry To Reveal How Cisplatin Treatment Modulates the Release of Catecholamine Transmitters during Exocytosis

The pretreatment of cultured pheochromocytoma (PC12) cells with cis-diamminedichloroplatinum (cisplatin), an anti-cancer drug, influences the exocytotic ability of the cells in a dose-dependent manner. Low concentrations of cisplatin stimulate catecholamine release whereas high concentrations inhibit it. Single-cell amperometry reflects that 2 μm cisplatin treatment increases the frequency of exocytotic events and reduces their duration, whereas 100 μm cisplatin treatment decreases the frequency of exocytotic events and increases their duration. Furthermore, the stability of the initial fusion pore that is formed in the lipid membrane during exocytosis is also regulated differentially by different cisplatin concentrations. This study thus suggests that cisplatin influences exocytosis by multiple mechanisms.