Hypericin Photodynamic Therapy Induces Cytotoxicity and Modulates Cytokine Secretion in MCF-7 Breast Cancer Cells

Abstract

Background/Aim: Photodynamic therapy uses a photosensitizer and light to generate reactive oxygen species that kill tumor cells and can shift inflammatory signaling. Hypericin is a potent photosensitizer, but its immunomodulatory impact in breast cancer needs clarification. We evaluated the phototoxic and cytokine-modulating effects of hypericin-mediated photodynamic therapy in MCF-7 human breast adenocarcinoma cells. This study examines how HYP-PDT affects MCF-7 breast cancer cells by assessing viability and cytokine secretion to guide the development of targeted, immune-enhancing PDT protocols. Methods: MCF-7 cells were incubated with hypericin at 0, 0.125, 0.25, 0.5, or 1 μM, then exposed to light doses of 0, 1, 2, or 5 J/cm². Viability was measured 24 h later by MTT; selected conditions were also assessed by Trypan Blue. Cell supernatants collected after sublethal treatment were analyzed for IL-6, IL-8, IL-10, and TNF-α using a multiplex immunoassay. Experiments were repeated four times. Statistical analyses followed the study's plan for group comparisons. Results: At 1 J/cm², MTT values did not differ from matched dark controls across hypericin concentrations. At 2 and 5 J/cm², some conditions showed increased MTT signal relative to controls, indicating higher metabolic activity; Trypan Blue performed at 0 J/cm² showed a concentration-dependent reduction in viability with hypericin. Hypericin-PDT decreased IL-6 and IL-8 concentrations and increased TNF-α in MCF-7 supernatants. No statistically significant changes were detected for IL-10. Conclusions: Hypericin-PDT altered inflammatory readouts in MCF-7 cells, with reductions in IL-6 and IL-8 and an increase in TNF-α, consistent with a pro-inflammatory shift. Viability results suggest condition-dependent changes in metabolic activity or survival effects that warrant confirmation with matched cell counts across all light doses. These findings support further standardized dosimetry and multi-line validation of hypericin-PDT in breast cancer models.

Keywords: IL-6; IL-8; MCF-7; TNF-α; breast cancer; cytokines; hypericin; photodynamic therapy.

Figure 1

MTT reduction [%] in the MCF-7 cell line for different Hypericin concentrations: 0 μM, 0.125 μM, 0.25 μM, 0.5 μM, and 1 μM, and light dose (A) 0 J/cm²; (B) 1 J/cm²; (C) 2 J/cm²; and (D) 5 J/cm². The values represent the means ± SD. (A) p > 0.05. (B) ** p < 0.05; (C) p > 0.05. (D) p > 0.05. In the study, the reduction of MTT was calculated as a percentage of the dark group control for each cell line in %.

Figure 2

The concentration of IL-6 and IL-8 in the MCF-7 cell line. Both figures are the results of various light doses (0 J/cm², 1 J/cm², 2 J/cm², 5 J/cm²) and the results of various Hypericin concentrations (0 μM, 0.125 μM, 0.25 μM, 0.5 μM). The values represent the means ± SD.

Figure 3

The concentration of IL-10 in the MCF-7 cell line and the concentration of TNF-α in the MCF-7 cell line. The values represent the means ± SD.

Figure 4

Dose response of MTT signal across hypericin concentrations and light doses. The 50% effect level was not crossed. IC50 not reached within the tested range.

Figure 5

PDT-induced effect of the HY on MCF-7 cells. Light scattering microscopy images show the time-dependent response of the cells to treatment. The dimension of the area displayed is 450 µm × 400 µm. These are not overlay images. Bar scale = 50 µm. (A) Control cells before exposure to the device, showing normal morphology. (B) Cells after 1 h of exposure, maintaining mostly intact structure with slight surface changes. (C) Cells after 3 h of exposure, showing increased darkening and early signs of cell damage. (D) Cells after 24 h of exposure, displaying extensive structural degradation and reduced cell density.

Figure 6

Location of HY into MCF-7 cells after treatment for 3 h: (A) fluorescence of HY; (B) fluorescence of HY after treatment for 24 h. Magnification 100×. Scale bar = 50 μm. We expect the observed diffraction of light to be the spreading out of waves as they reach and pass through an aperture or around objects. It occurs when the size of the aperture or obstacle is of the same order of magnitude as the wavelength of the incident wave.

Figure 7

Hypericin photodynamic mechanism.

Figure 8

Uptake of Hypericin in the MCF-7 cells in %. The calibration was performed base signal intensity. We consider that the stock solution has a maximum of 100% intensity.