Chemotherapeutic Effect of Calcium Sulfide Nanostructures in non‐small cell lung carcinoma by intrinsic and extrinsic apoptotic pathways
Authors: Rosado Maria Milagros Figueroa , Forti Kevin Muñoz , Martínez Gerardo Arroyo , Carattini Thalia Vega , Rivera Abigail Ruiz , Santiago Pedro , Castro Miguel E , Suárez Edu 
Published: November 2023

Apoptosis or programed cell death is characterized by metabolic changes and energy‐dependent mechanisms aimed to ensure cell viability and proper function. A failure in the activation of pro‐apoptotic processes is considered a major contributor to the survival of tumor cells, induction of malignant transformation, tumor growth and subsequently, cancer as a disease. The apoptotic cascade can be triggered by a variety of internal and external stimuli, the results of which are classified into two signaling pathways: Extrinsic and Intrinsic, based on the origin of the stimuli and the mediators involved. The principal mediators are caspases‐8 and ‐9, which are responsible for a subtype of apoptosis known as Caspase dependent apoptosis. Previous studies in our laboratory showed that non‐small lung cell carcinoma cells (NSCLC) (HCC827) treated with proprietary calcium sulfide nanostructures (CaS) exhibited induction of apoptosis starting at 48 hours after treatment (p<0.05). In the new stage of this project, we aimed to determine the expression of apoptotic factors (Bax, Caspase‐3, Casapse‐8), the electron transport chain component Cytochrome C (CytoC), and the metabolic enzyme GAPDH, 24‐, 48‐ and 72‐hours after a single dose addition of CaS nanostructures. We hypothesized that CaS nanostructures treatment in HCC827 cells will selectively activate the intrinsic and extrinsic pathways; increasing the cytosolic CytoC expression and decreasing the expression of GADPH when compared to non‐malignant MRC5 cells. The HCC827 and MRC‐5 cells were treated with DMSO [1%] (vehicle control), etoposide [10μM] (positive control), and CaS nanostructures [3.8μM] (experimental treatment) at 0‐hours. To study the target proteins, we performed Western Blot analysis, densitometry and statistical analyses to evaluate changes in expression of the studied proteins. Our preliminary results showed that Bax expression decreased through time in the MRC‐5 cells treated with CaS compared to DMSO; while its expression increased in the HCC827 cells at 48 hours (p<0.05). At 24 hours, the expression of caspases‐8 and ‐3 in HCC827 increased when compared to DMSO, but it was not statistically significant. In MRC‐5 cells, caspase‐8 was observed at 24 hours. In contrast, caspase‐3 expression increased compared to DMSO and remained constantly increased at 24‐ and 48‐ hours. The expression of CytoC was observed in the HCC827 cells after 48 hours of exposure to CaS nanostructures; while it was not detected in MRC‐5 at any time point. These results suggest that CaS structures have a pro‐apoptotic effect, involving both intrinsic and extrinsic pathways, accepting this part of the hypothesis. As well CytoC was detected only in HCC827, suggesting release from the mitochondria that would promote the intrinsic apoptotic pathway. However, our hypothesis was not accepted for the GADPH protein expression, by obtaining the opposite results. This may be explained as an attempt of the malignant cells to survive. We are currently studying additional pro‐apoptotic factors, such as caspase‐9 and caspase‐7, to elucidate the mechanism CaS nanostructures within these pathways, which may represent a selective chemotherapeutic agent for NSCLC.

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