Cell viability was assessed at different time points (3, 6, 12, and 24 hours) following treatment with ALA-PDT and HCQ alone or in combination. The CCK-8 assay results demonstrated that cell viability was significantly decreased in the group treated with ALA-PDT + HCQ compared to the other three groups (Figure 1a). To investigate the role of HCQ in this effect, we performed flow cytometry analysis of cellular ROS levels. Interestingly, there was no significant difference in ROS levels between the control, HCQ alone, and ALA-PDT alone groups (Figures 1b and c), while a significant increase in ROS production was observed following exposure to ALA-PDT + HCQ. To determine whether this increase in ROS levels contributed to the observed decrease in cell viability, HeLa cells were co-treated with NAC. Results showed that while ALA-PDT + HCQ significantly inhibited cell proliferation, this effect was partially reversed by NAC treatment. Specifically, cell viability was higher in cells treated with ALA-PDT + HCQ + NAC than those treated with ALA-PDT alone; however, this change did not reach statistical significance. Taken together, these findings suggest that HCQ enhances the antitumor effect of ALA-PDT by increasing ROS production in HeLa cells. Moreover, our results provide important insights for future development of combination therapies utilizing ALA-PDT and HCQ for cancer treatment.
