Pllans-II: Unveiling the Action Mechanism of a Promising Chemotherapeutic Agent Targeting Cervical Cancer Cell Adhesion and Survival Pathways.

Despite advances in chemotherapeutic drugs used against cervical cancer, available chemotherapy treatments adversely affect the patient's quality of life. For this reason, new molecules from natural sources with antitumor potential and few side effects are required. In previous research, Pllans-II, a phospholipase A2 type-Asp49 from Porthidium lansbergii lansbergii snake venom, has shown selective attack against the HeLa and Ca Ski cervical cancer cell lines. This work suggests that the cytotoxic effect generated by Pllans-II on HeLa cells is triggered without affecting the integrity of the cytoplasmic membrane or depolarizing the mitochondrial membranes. The results allow us to establish that cell death in HeLa is related to the junction blockage between α5ß1 integrins and fibronectin of the extracellular matrix. Pllans-II reduces the cells' ability of adhesion and affects survival and proliferation pathways mediated by intracellular communication with the external environment. Our findings confirmed Pllans-II as a potential prototype for developing a selective chemotherapeutic drug against cervical cancer.

Exploring the Safety of Pllans-II and Antitumoral Potential of Its Recombinant Isoform in Cervical Cancer Therapy.

The antitumor potential of proteins from snake venoms has been studied in recent decades, and evidence has emerged that phospholipases A2 can selectively attack cells of various types of tumors. Previous results have shown that phospholipase A2 "Pllans-II," isolated from Porthidium lansbergii lansbergii snake venom, displayed antitumoral activity on cervical cancer and did not alter the viability of non-tumorigenic cells. However, until now, there was no evidence of its safety at the local and systemic levels, nor had experiments been developed to demonstrate that its production using recombinant technology allows us to obtain a molecule with effects similar to those generated by native phospholipase. Thus, we evaluated the impact caused by Pllans-II on murine biomodels, determining whether it induced local hemorrhage or increased pro-inflammatory and liver damage markers and histological alterations in the liver and kidneys. Additionally, the protein was produced using recombinant technology using a pET28a expression vector and the BL21 (DE3) Escherichia coli strain. Equally, its enzymatic activity and anticancer effect were evaluated on cervical cancer lines such as HeLa and Ca Ski. The results demonstrated that Pllans-II did not generate hemorrhagic activity, nor did it increase the pro-inflammatory cytokines IL-6, IL-1B, or TNF-α at doses of 3.28, 1.64, and 0.82 mg/kg. There was also no evidence of organ damage, and only ALT and AST increased in mild levels at the two highest concentrations. Additionally, the recombinant version of Pllans-II showed conservation in its catalytic activity and the ability to generate death in HeLa and Ca Ski cells (42% and 23%, respectively). These results demonstrate the innocuity of Pllans-II at the lowest dose and constitute an advance in considering a molecule produced using recombinant technology a drug candidate for selective attacks against cervical cancer.

Proteomic and functional analyses of Lachesis acrochorda snake venom from the Valle del Cauca Department of Colombia.

Lachesis acrochorda envenomation has a lethality rate of approximately 90%. Despite its high lethality, little is known about its local and systemic effects and its relationship with its protein content. Thus, to increase knowledge of L. acrochorda snake venom from the Southwestern ecoregion of Colombia, we developed a proteomic analysis using a "bottom-up shotgun proteomic profiling" approach. Besides, we evaluated toxinological properties and compared the effects with the Bothrops asper snake venom activities. The RP-HPLC profile showed similarities with the L. acrochorda snake venom from the Northwestern ecoregion of Colombia. However, the results displayed differences in the protein families identified, probably due to the proteomic identification strategy. The in vitro and in vivo tests showed a L. acrochorda snake venom with Phospholipase A2 and metalloproteinase activities related to myotoxic, edematic, and hemorrhagic effects. Nevertheless, the L. acrochorda snake venom displayed a low lethality despite a large amount of inoculated venom. This investigation's results will help us improve the knowledge about the relationship between the clinical manifestations of L. acrochorda envenomation and the venom protein content.

Pllans-II Induces Cell Death in Cervical Cancer Squamous Epithelial Cells via Unfolded Protein Accumulation and Endoplasmic Reticulum Stress.

Due to the lack of chemotherapeutic drugs that selectively affect cervical cancer cells, natural sources such as snake venom are currently being investigated for molecules with antitumor potential. Pllans-II, a phospholipase A2 type-Asp49 from Porthidium lansbergii lansbergii snake venom, induced cell death in a cervical cancer cell line-Ca Ski-related to dysfunction in the ability to resolve endoplasmic reticulum stress, evidenced by sub-expression of genes such as PERK, ERO1 PDIs, HSP70, and CHOP. Western blot analysis validated the last two genes' sub-expression at the protein level. In addition, Pllans-II presented a dose-dependent cytotoxic effect on cancer cells and an insignificant effect on healthy endothelial cells (HUVEC). Additionally, Pllans-II inhibited cancer cells' adhesion and migration capacity, induced cell cycle arrest in the G2/M phase, and induced apoptosis stimulated possibly by the extrinsic route. These results demonstrate for the first time that Pllans-II has an antitumor effect on a squamous epithelial cervical cancer cell line and represents a possible biotechnological tool for designing a prominent antitumor agent.

First functional and proteomic analysis of Bothrops asper snake venom from Gorgona Island - Colombia, and its comparative characterization with two Colombian Southwest ecoregions.

Bothrops asper envenoming is a public health problem in tropical regions of Latin America. Bothrops asper has spread until Gorgona Island in the Pacific Colombian Ocean, but its biochemical venom characterization is poorly known. Thus, to increase knowledge on Bothrops species venoms, we developed for the first time the proteomic analysis using a shotgun approach and performed functional evaluations relevant to its toxicity and compared with two Colombian Southwest ecoregions from the Pacific and Western sides. Besides, we evaluated two antivenoms produced in Colombia (INS and PROBIOL) against three B. asper venom ecoregions through the ELISA approach and first-generation antivenom against B. asper from Gorgona Island. The protein components of B. asper from Gorgona Island were assigned to nine known protein families, sharing a conserved compositional pattern with B. asper from the pacific ecoregion. The RP-HPLC and in vitro activity suggest a phenotypic congruence in the expression of PLA2s and metalloproteinases between the B. asper snake venom from Gorgona Island and pacific, but inversely to the Western ecoregion. Additionally, the antivenoms immunoreactivity against the three B. asper lineage venoms was different. The INS displayed higher titers than PROBIOL against all the venoms and exhibited the most effective immunocapturing capacity against the individual components of snake venom from Gorgona Island. The results of this investigation suggest that B. asper from Gorgona Island displayed similar clinical manifestations concerning the Pacific ecoregion, and the immunoreactivity by antivenoms could be used after B. asper envenomation in Gorgona Island, using one of them preferably.

Individual variations in the protein profiles and functional activities of the eyelash palm pit-viper (Bothriechis schlegelii) venom from the Colombian southwest region.

Bothriechis schlegelii is a venomous snake found in Central and South America, mainly sighted in regions devoted to agriculture. However, in Colombia, little is known about its contribution to the total envenoming cases. Furthermore, there are no reports of the biochemical and functional activities of venoms from the southwest populations, and the differences respecting other populations are unknown. This study analyzed the protein profiles of venom samples obtained from three specimens originating from this region of Colombia using electrophoresis and chromatography. The lethality, edema-induction, hemorrhagic, defibrinating, coagulant, and indirect hemolytic activities were also evaluated. As a result, venoms were composed of proteins with a wide range of molecular weights, most of them below <37 kDa, with differences between male and female electrophoretic and chromatographic profiles. These variations were also observed in the evaluation of venom functional activities such as pro-coagulant, indirect hemolytic, and edema-inducing activities, whereas neither hemorrhagic nor defibrinating activities were detected. These results are also different considering reports with venom samples from other geographical locations, restating the existence of high intraspecific variability in B. schlegelii venoms, which could have relevant pathophysiological and therapeutic implications.

Evaluation of the systemic alterations triggers by Porthidium lansbergii lansbergii snake venom.

The systemic effects generated by Porthidium lansbergii lansbergii envenoming, a species found in the northern region of Colombia, is poorly known. The present study aimed to analyze for the first time the mice's behavior, the histological alterations, and changes in biochemical markers levels resulting from the intraperitoneal injection of an LD50 of P. lansbergii lansbergii snake venom on mice. The envenoming mice displayed hypodynamic condition, clonic head movements, accompanied by bradypnea and thoracoabdominal imbalance. After 7 h of envenoming, the mice showed an ecchymotic region at the injection site, including bleeding in the pleural, liver, and kidney capsules. The effect on the brain revealed a micro-hemorrhage in the sensorimotor cortex with substantial loss of neurons. The venom caused dilated blood vessels in lung tissue, with endothelial necrosis associated with alveolar rupture. The liver showed parenchyma alteration with many extravasated erythrocytes. The kidneys exhibited renal tubules necrosis and a statistically significant increase in creatinine concentration. ALP and ALT's enzymatic activities remained constant at 7 h after envenoming but increased at 12 h. AST and LDH were significantly increased at 7 h but decreased to the near baseline 12 h after venom administration. Massive hemorrhages could trigger a hypovolemic shock, which could lead to death after several h without treatment. Knowledge of P. lansbergii lansbergii snake bites' injuries is essential to make the appropriate diagnostic in human envenoming cases by this snake.

Cervical cancer and potential pharmacological treatment with snake venoms.

Cervical cancer is the fourth most common cancer worldwide in women. Apoptosis reactivation has become the main strategy for decreasing cancer proliferation. There is a need to extend the search for new drugs to implement more effective and less toxic strategies for cervical cancer treatment. Research has been carried out to find new drugs that have minimal side effects and that focus on the tumor microenvironment, particularly in the induction of cellular apoptosis and cell migration and the inhibition of angiogenesis. Potent toxins from snake venoms have shown potential as sources for the synthesis of new drugs with such characteristics. The present work aimed to describe cervical cancer characteristics, associated risk factors, current treatments and to highlight the effects of toxins isolated from the venom of snakes of the Viperidae family on cervical cancer cell lines.