Cytotoxic Effects of Combinative ZnPcS4 Photosensitizer Photodynamic Therapy (PDT) and Cannabidiol (CBD) on a Cervical Cancer Cell Line

Abstract

The most prevalent type of gynecological malignancy globally is cervical cancer (CC). Complicated by tumor resistance and metastasis, it remains the leading cause of cancer deaths in women in South Africa. Early CC is managed by hysterectomy, chemotherapy, radiation, and more recently, immunotherapy. Although these treatments provide clinical benefits, many patients experience adverse effects and secondary CC spread. To minimize this, novel and innovative treatment methods need to be investigated. Photodynamic therapy (PDT) is an advantageous treatment modality that is non-invasive, with limited side effects. The Cannabis sativa L. plant isolate, cannabidiol (CBD), has anti-cancer effects, which inhibit tumor growth and spread. This study investigated the cytotoxic combinative effect of PDT and CBD on CC HeLa cells. The effects were assessed by exposing in vitro HeLa CC-cultured cells to varying doses of ZnPcS₄ photosensitizer (PS) PDT and CBD, with a fluency of 10 J/cm² and 673 nm irradiation. HeLa CC cells, which received the predetermined lowest dose concentrations (ICD₅₀) of 0.125 µM ZnPcS₄ PS plus 0.5 µM CBD to yield 50% cytotoxicity post-laser irradiation, reported highly significant and advantageous forms of cell death. Flow cytometry cell death pathway quantitative analysis showed that only 13% of HeLa cells were found to be viable, 7% were in early apoptosis and 64% were in late favorable forms of apoptotic cell death, with a minor 16% of necrosis post-PDT. Findings suggest that this combined treatment approach can possibly induce primary cellular destruction, as well as limit CC metastatic spread, and so warrants further investigation.

Keywords: HeLa; ZnPcS4; cannabidiol (CBD); cervical cancer; photodynamic therapy (PDT); photosensitizer; phthalocyanines.

Figure 1

LDH membrane integrity response of HeLa cells treated with varying increasing concentrations of ZnPcS₄ PS, demonstrating a significant dose-dependent increase in cellular cytotoxicity post PDT (p < 0.01** or p < 0.001***).

Figure 2

LDH integrity response of HeLa cells treated with varying increasing concentrations of CBD, demonstrating a significant dose-dependent increase in cellular cytotoxicity pre- and post-irradiation (p < 0.01** or p < 0.001***).

Figure 3

Subcellular localization comparison of various control and experimental groups of HeLa cells treated with singular and combinative ZnPcS₄ PS and/or CBD pre-irradiation; DAPI-stained nuclei (blue), ICAM-1 cellular membrane proteins (green) and Cy5 fluorescence from ZnPcS₄ PS subcellular localization (red) (40× magnification, 50 µM scale bar).

Figure 4

Subcellular localization comparison of various control and experimental groups of WS1 normal fibroblast cells, treated with singular and combinative ZnPcS₄ PS and/or CBD pre-irradiation; DAPI-stained nuclei (blue), ICAM-1 cellular membrane proteins (green), and Cy5 fluorescence from ZnPcS₄ PS subcellular localization (red) (40× magnification, 50 µM scale bar).

Figure 5

Percentage of different stages of cell death using the flow cytometry Annexin V-FITC/PI staining method on various HeLa control and experimental groups, within ZnPcS₄ PS and CBD PDT/irradiation combinative assays (p < 0.05*, p < 0.01** or p < 0.001***).

Figure 6

Percentage of different stages of cell death using the flow cytometry Annexin V-FITC/PI staining method on various WS1 normal fibroblast control and experimental groups, within ZnPcS₄ PS and CBD PDT/irradiation combinative assays.

Figure 7

Percentage of cell proliferation among various HeLa control and experimental groups, within ZnPcS₄ PS and CBD post-irradiation combinative assays (p < 0.001***).

Figure 8

Percentage of cell proliferation of various WS1 normal fibroblast control and experimental groups, within ZnPcS₄ PS and CBD post-irradiation combinative assays.

Figure 9

(a) Chemical structure of CBD. (b) Chemical structure of ZnPcS₄ PS.