Cytotoxic effect of different treatment parameters in pressurized intraperitoneal aerosol chemotherapy (PIPAC) on the in vitro proliferation of human colonic cancer cells.

BACKGROUND: Pressurized intraperitoneal aerosol chemotherapy (PIPAC) has been recently reported as a new approach for intraperitoneal chemotherapy (IPC). By means of a patented micropump, the liquid chemotherapy is delivered into the peritoneal cavity as an aerosol which is supposed to achieve "gas-like" distribution. However, recent data report that the fraction of the submicron aerosol (gas-like) is less than 3 vol% of the total amount of aerosolized chemotherapy. Until today, possible modifications of treatment parameters during PIPAC with the aim of improving therapeutic outcomes have not been studied yet. This study aims to establish an in vitro PIPAC model to explore the cytotoxic effect of the submicron aerosol fraction and to investigate the impact of different application parameters on the cytotoxic effect of PIPAC on human colonic cancer cells. METHODS: An in vitro model using HCT8 colon adenocarcinoma wild-type cells (HCT8WT) and multi-chemotherapy refractory subline (HCT8RT) was established. Different experimental parameters such as pressure, drug dosage, time exposure, and system temperature were monitored in order to search for the conditions with a higher impact on cell toxicity. Cell proliferation was determined by means of colorimetric MTT assay 48 h following PIPAC exposures. RESULTS: Standard operational parameters applied for PIPAC therapy depicted a cytotoxic effect of the submicron aerosol fraction generated by the PIPAC micropump. We also observed that increasing pressure significantly enhanced tumor cell toxicity in both wild-type and chemotherapy-resistant cells. A maximum of cytotoxicity was observed at 15 mmHg. Pressure >15 mmHg did not show additional cytotoxic effect on cells. Increased oxaliplatin dosage resulted in progressively higher cell toxicity as expected. However, in resistant cells, a significant effect was only found at higher drug concentrations. Neither an extension of exposure time nor an increase in temperature of the aerosolized chemotherapy solution added an improvement in cytotoxicity. CONCLUSIONS: In this in vitro PIPAC model, the gas-like PIPAC aerosol fraction showed a cytotoxic effect which was enhanced by higher intra-abdominal pressure with a maximum at 15 mmHg. Similar findings were observed for drug dose escalation. A phase I dose escalation study is currently performed at our institution. However, increasing the intra-abdominal pressure might be a first and simple way to enhance the cytotoxic effect of PIPAC therapy which needs further clinical investigations.

Therapeutic potential of antiviral drugs targeting chemorefractory colorectal adenocarcinoma cells overexpressing endogenous retroviral elements.

BACKGROUND: Endoretroviruses account for circa 8 % of all transposable elements found in the genome of humans and other animals. They represent a genetic footprint of ancestral germ-cell infections of exoviruses that is transmittable to the progeny by Mendelian segregation. Traces of human endogenous retroviruses are physiologically expressed in ovarial, testicular and placental tissues as well as in stem cells. In addition, a number of these fossil viral elements have also been related to carcinogenesis. However, a relation between endoretroviruses expression and chemoresistance has not been reported yet. METHODS: Twenty colorectal carcinoma patient samples were scrutinized for HERV-WE1 and HERV-FRD1 endoretroviruses using immunohistochemical approaches. In order to search for differential expression of these elements in chemotherapy refractory cells, a resistant HCT8 colon carcinoma subline was developed by serial etoposide exposure. Endoretroviral elements were detected by immunocytochemical staining, qPCR and ELISA. IC50-values of antiviral and cytostatic drugs in HCT8 cells were determined by MTT proliferation assay. The antivirals-cytostatics interaction was evaluated by the isobologram method. RESULTS: In this work, we show for the first time that HERV-WE1, HERV-FRD1, HERV-31, and HERV-V1 are a) simultaneously expressed in treatment-naïve colon carcinoma cells and b) upregulated after cytostatic exposure, suggesting that these retroviral elements are intimately related to chemotherapy resistance. We found a number of antiviral drugs to have cytotoxic activity and the ability to force the downregulation of HERV proteins in vitro. We also demonstrate that the use of different antiviral compounds alone or in combination with anticancer agents results in a synergistic antiproliferative effect and downregulation of different endoretroviral elements in highly chemotherapy-resistant colorectal tumor cells. CONCLUSIONS: Enhanced HERV-expression is associated with chemoresistance in colon carcinomas which can be overcome by antiviral drugs alone or in combination with anticancer drugs. Therefore, the introduction of antiviral compounds to the current chemotherapy regimens potentially improves patient outcomes.

Atypical cell populations associated with acquired resistance to cytostatics and cancer stem cell features: the role of mitochondria in nuclear encapsulation.

Until recently, acquired resistance to cytostatics had mostly been attributed to biochemical mechanisms such as decreased intake and/or increased efflux of therapeutics, enhanced DNA repair, and altered activity or deregulation of target proteins. Although these mechanisms have been widely investigated, little is known about membrane barriers responsible for the chemical imperviousness of cell compartments and cellular segregation in cytostatic-treated tumors. In highly heterogeneous cross-resistant and radiorefractory cell populations selected by exposure to anticancer agents, we found a number of atypical recurrent cell types in (1) tumor cell cultures of different embryonic origins, (2) mouse xenografts, and (3) paraffin sections from patient tumors. Alongside morphologic peculiarities, these populations presented cancer stem cell markers, aberrant signaling pathways, and a set of deregulated miRNAs known to confer both stem-cell phenotypes and highly aggressive tumor behavior. The first type, named spiral cells, is marked by a spiral arrangement of nuclei. The second type, monastery cells, is characterized by prominent walls inside which daughter cells can be seen maturing amid a rich mitochondrial environment. The third type, called pregnant cells, is a giant cell with a syncytium-like morphology, a main nucleus, and many endoreplicative functional progeny cells. A rare fourth cell type identified in leukemia was christened shepherd cells, as it was always associated with clusters of smaller cells. Furthermore, a portion of resistant tumor cells displayed nuclear encapsulation via mitochondrial aggregation in the nuclear perimeter in response to cytostatic insults, probably conferring imperviousness to drugs and long periods of dormancy until nuclear eclosion takes place. This phenomenon was correlated with an increase in both intracellular and intercellular mitochondrial traffic as well as with the uptake of free extracellular mitochondria. All these cellular disorders could, in fact, be found in untreated tumor cells but were more pronounced in resistant entities, suggesting a natural mechanism of cell survival triggered by chemical injury, or a primitive strategy to ensure stemming, self-renewal, and differentiation under adverse conditions, a fact that may play a significant role in chemotherapy outcomes.

Identification of compounds that selectively target highly chemotherapy refractory neuroblastoma cancer stem cells.

Relapse of cancer months or years after an apparently successful therapy is probably caused by cancer stem cells (CSCs) due to their intrinsic features like dormant periods, radiorefraction, and acquired multidrug resistance (MDR) phenotypes, among other mechanisms of cellular drug evasiveness. Thus, the lack of currently efficacious interventions remains a major problem in the treatment of malignancies, together with the inability of existing drugs to destroy specifically CSCs. Neuroblastomas per se are highly chemotherapy-refractory extracranial tumors in infants with very low survival rates. So far, no effective cytostatics against this kind of tumors are clinically available. Therefore, we have put much effort into the development of agents to efficiently combat this malignancy. For this purpose, we tested several compounds isolated from Cuban propolis on induced CSCs (iCSC) derived from LAN-1 neuroblastoma cells which expressed several characteristics of tumor-initiating cells both in in-vitro and in-vivo models. Some small molecules such as flavonoids and polycyclic polyprenylated acylphloroglucinols (PPAP) were isolated using successive RT-HPLC cycles and identified employing mass spectrometry and NMR spectroscopic techniques. Their cytotoxicity was first screened in sensitive cell systems by MTT proliferation assays and afterwards studied in less sensitive neuroblastoma iCSC models. We found several compounds with considerable anti-iCSC activity, most of them belonging to the PPAP class. The majority of the compounds act in a pleiotropic manner on the molecular biology of tumors although their specific targets remain unclear. Nevertheless, two substances, one of them a flavonoid, induced a strong disruption of tubulin polymerization. In addition, an unknown compound strongly inhibited replicative enzymes like toposimerases I/II and DNA polymerase. Here, we report for the first time cytotoxic activities of small molecules isolated from Caribbean propolis which could be promising therapeutics or lead structures against therapy-refractory neuroblastoma entities. *Contributed equally.

7-epi-nemorosone from Clusia rosea induces apoptosis, androgen receptor down-regulation and dysregulation of PSA levels in LNCaP prostate carcinoma cells.

The aim of this work was to characterize the antitumoral activity of the plant compound 7-epi-nemorosone in prostate carcinoma cell lines. Prostate cancer is the most frequently diagnosed malignancy and the second-leading cause of cancer death in men. In spite of the current therapeutic options for this cancer entity, many patients die due to metastases in distant organs and acquired chemotherapy resistance. Thus, approaches to provide improvements in outcome and quality of life for such patients are urgently needed. Recently, the polyisoprenylated benzophenone 7-epi-nemorosone, originally collected by honeybees from Clusia rosea and Clusia grandiflora (Clusiaceae), has been described to be a potent antitumoral agent. Here, its activity in prostate carcinoma is reported. 7-epi-nemorosone was isolated from Caribbean propolis employing RP-HPLC techniques. Its cytotoxicity was assessed using the MTT proliferation assay in human androgen-dependent prostate carcinoma LNCaP cells including an MDR1(+) sub-line. No cross-resistance was detected. FACS-based cell cycle analysis revealed a significant increase in the sub-G0/G1, G1, and depletion in the S phase populations. A concomitant down-regulation of cyclins D1/D3 and CDK 4/6 in LNCaP cells was detected by Western blot. Annexin-V-FITC labeling and caspase-3 cleavage assays showed that 7-epi-nemorosone induced apoptotic events. Major signal transduction elements such as p38 MAPK and Akt/PKB as well as androgen receptor AR and PSA production were found to be down-regulated after exposure to the drug. ERK1/2 protein levels and phosphorylation status were down-regulated accompanied by up-regulation but inhibition of the activity of their immediate upstream kinases MEK1/2. Additionally, Akt/PKB enzymatic activity was effectively inhibited at a similar concentration as for MEK1/2. Here, we demonstrate for the first time that 7-epi-nemorosone exerts cytotoxicity in an androgen-dependent prostate carcinoma entity by targeting the MEK1/2 signal transducer.