SA3 - pH-Responsive ZIF-8 Precisely Induces Apoptosis of Oral Squamous Cell Carcinoma Over Orofacial Mesenchymal Stem/Stromal Cells

Abstract: The pH-responsive zeolite imidazolate framework-8 (ZIF-8) has recently gained attention as a drug delivery system for conventional cancer chemotherapeutics.¹ ZIF-8 may degrade in low pH conditions, such as the acidic tumor microenvironment (PH = 5.5), while remaining stable under physiological pH (PH = 7.4). In our previous study, ZIF-8 was shown to be capable of absorbing and releasing 5-fluorouracil (5-FU) in a controlled manner. Through in vitro cell biocompatibility studies, 5-FU incorporated ZIF-8 inhibited oral cancer cell line SCC7 in a synergistic manner,² which indicated that ZIF-8 not only acts as a carrier to deliver anti-cancer drug but can also induce cancer cell apoptosis. Since oral cancer resides in transformed epithelium and interacts with surrounding stroma,³ the present study aims to observe how ZIF-8 affects orofacial mesenchymal stem/stromal cells (OMSC), which is crucial to understanding its holistic impact towards the body. We hypothesized that ZIF-8 decreases cell viability and enhances apoptosis in OSCC cell lines but has no significant effect on the cell viability and apoptosis of primary isolated OMSC.

ZIF-8 was purchased from Sigma Aldrich and heat activated for 24 h at 160℃. SCC7 oral cancer cell line was cultured in alpha MEM supplemented with 10% (v/v) FBS, 1% (v/v) 200 mM glutamate, and 1% (v/v) penicillin/streptomycin. PDLSC and DPSC were obtained from healthy patients undergoing third molar extraction as discarded biological sample at the University of Pennsylvania School of Dental Medicine as guided by the approved institutional review board (IRB) guidelines. The effects of ZIF-8 on SCC7 and OMSC (including PDLSC and DPSC) were compared through nuclear staining, live/dead staining, and immunocytochemistry. SCC7, PDLSC and DPSC were exposed to 0, 1, 10, and 100 μg/mL ZIF-8 and treated with toluidine blue nuclear stain. Based on these results, the LIVE/DEAD Viability/Cytotoxicity protocol was applied to SCC7 and DPSC with 0 and 100 μg/mL ZIF-8. Finally, SCC7 and DPSC were exposed to 100 μg/mL ZIF-8 and stained with an immunocytochemistry protocol that included bax, caspase 3, caspase 6, and caspase 10 antibodies.

Cells with chemical or immunofluorescent staining were imaged by Olympus IX71 microscope with digital camera. Comparisons between two groups were analyzed by independent unpaired two-tailed Student’s t test, and the comparisons between more than two groups were analyzed by one-way ANOVA with the Bonferroni adjustment. P values < .05 were considered statistically significant.

Nuclear staining demonstrated significantly lower SCC7 viability than OMSCs at 100 μg/mL ZIF-8 [Fig 1]; therefore, this concentration was selected for subsequent experiments. While the proportion of nonviable DPSCs remained similar between 100 μg/mL (34.25% ± 24.53%) and control (30.97% ± 11.31%), the proportion of nonviable SCC7s was significantly higher at 100 μg/mL (98.14% ± 3.09%) than control (45.95% ± 10.79%) [Fig 2]. Immunochemistry also showed higher levels of apoptotic proteins for SCC7 than DPSC at 100 μg/mL ZIF-8 [Fig 3].

ZIF-8 not only serves as a drug delivery system, but also holds significant promise by itself as a targeted therapeutic agent for oral cancer, showing a selective cytotoxic effect on cancerous SCC7 cells while sparing the viability of OMSC populations. Collectively, ZIF-8 at 100 μg/mL has an inhibitory effect towards SCC7, but no significant effect on the viability and apoptosis of OMSC.

References:
1. Hao J, Stavljenić Milašin I, Batu Eken Z, Mravak-Stipetic M, Pavelić K, Ozer F. Effects of Zeolite as a Drug Delivery System on Cancer Therapy: A Systematic Review. Molecules. 2021 Oct 14;26(20):6196.
2. Hao J, Chen C, Pavelic K, Ozer F. ZIF-8 as a pH-Responsive Nanoplatform for 5-Fluorouracil Delivery in the Chemotherapy of Oral Squamous Cell Carcinoma. Int J Mol Sci. 2024 Aug 27;25(17):9292.
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