CC BY
9DOI 10.24412/cl-37136-2023-1-58-62
PHOTODYNAMIC THERAPY ENHANCES THE BERBERINE INDUCED CYTOTOXICITY IN
LUNG CANCER CELLS
PAROMITA SARBADHIKARY1, BLASSAN P GEORGE1 AND HEIDI ABRAHAMSE1
Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, South Africa
paromitas@uj .ac.za
ABSTRACT
Even with a thorough understanding of the molecular causes of cancer and enormous strides in cancer therapy and diagnosis, cancer still poses a serious health risk and it is the second leading cause of death worldwide. According to the GLOBOCON report 2020, lung cancer continues to be the second most common cancer diagnosed, with an estimated 2.3 million new cases worldwide. According to reports, the global incidence rate of lung cancer in people of both sexes is 11.4% across all other cancer types with the highest mortality rate of 18% (1). Additionally, lung cancer is the deadliest disease due to its high metastatic potential, whereby nearly 70% of patients start receiving their diagnosis and treatment in advanced stages III or IV of the disease (2). Phytochemicals present a promising alternative to conventional therapies for improving treatment effectiveness in lung cancer patients, by overcoming the drawbacks of severe induced side effects and induction of resistance [3].
Among several different potent and safer antineoplastic phytochemicals, Berberine (BBR) (Fig. 1) is a phytochemical that has been used in traditional medicine with promising antitumor activity by inhibiting cell proliferation, angiogenesis, metastasis and inducing apoptosis by regulating different signaling pathways [4]. Moreover, BBR is reported to be a naturally occurring phototoxic alkaloid, which can be activated by near-ultraviolet light (NUV) and far-UV (FUV) to generate Reactive Oxygen Species (ROS) to induce a photosensitizing effect in cancer cells [5,6]. However, due to its poor aqueous solubility, very low bioavailability and membrane permeability, BBR can only induce its chemotoxic potential at very high concentrations [4,7]. However, combining the chemotoxic potential with the toxic effect of photogenerated ROS during photodynamic therapy (PDT) has the potential to induce strong phototoxic effects even at lower BBR concentrations. The purpose of this study is to evaluate the potential of BBR as a synergic chemo and phototoxic agent and observe the effects produced by this combination in lung carcinoma cells.
Figure 1: Chemical Structure of Berberine Chloride
The human epithelial lung cancer cells A549 (ATCC® CCL-185TM) obtained from American Type Culture Collection (ATCC) was maintained in Roswell Park Memorial Institute 1640 medium (RPMI) containing 1% antibiotics (penicillin/streptomycin) and 10% fetal bovine serum (FBS). The cells were grown at 37 °C in an 85% humified incubator under 5% CO2. For assessment of the chemotoxic potential of BBR, A549
cells were plated in 96 well plate and treated with BBR concentrations range from 2.5 to 320 |iM for 24 h. BBR induced phototoxicity and/or combined cytotoxicity was evaluated by treating the cells with a BBR concentration range of 2.5 to 63 |iM for 24 h. Following this, cells were irradiated with a 405 nm blue light semiconductor laser diode at light dose of 2 J/cm2. 24 h post treatment and irradiation the BBR induced chemotoxicity, phototoxicity and/or combined cytotoxicity was determined using Adenosine Triphosphate Assay (ATP) quantification assay using CellTiter-Glo® 3D Cell Viability Kit (Promega G9681). Briefly, following the treatment period, cells were washed twice with Hank's Balanced Salt Solution (HBSS). Following this, 50 |iL ATP reagent was added in each well containing 50 |iL of HBBS then mixed thoroughly and incubated at room temperature for 25 min. Post-incubation, the resultant ATP luminescence was recorded using a plate reader PerkinElmer, VICTOR NivoTM. The percentage viability for experimental groups was calculated with respect to the luminescence value in the control untreated group. Cellular damage post treatment was visualized by phase contrast inverted light microscope (Wirsam, Olympus CKX 41) attached to a digital camera (Olympus C5060-ADUS) at 200X magnification. The qualitative analysis of cell viability following treatment was carried out by staining the cells with a LIVE/DEAD cytotoxicity kit (Invitrogen L3224). All the experiments were performed in triplicates and independently repeated thrice. SigmaPlot version 14.0 software was used to analyze the mean and standard error (SE) values for each experimental group. Data points are represented as mean ± standard error (SE), and the difference between the control and experimental groups were statistically analyzed by Student's t-test, with a 95% confidence interval.
Fig. 2 shows the chemotoxic effect of BBR on the cell viability of A549 lung cancer cells treated for 24 h. Results showed BBR induced cytotoxicity in a concentration-dependent manner (Fig. 2 (a)). The IC50 value determined from the sigmoidal concentration-response curve is 63 |iM (Fig. 2 (b)). (a) (b)
Cone, of BBR (|iM) Log Cone, of BBR (MM)
Figure 2: (a) Cytotoxicity induced by different concentrations (0, 2.5,5, 10, 20, 40, 80 160 and 320 M) of Berberine (BBR) in A549 lung cancer cells treated for 24 h and determined by ATP proliferation assay, (b) sigmoidal concentration-response curve of BBR treatment. Data points in the graphs are represented as mean ±
standard errors from experiments repeated in triplicates. *(p <0.05), **(p <0.01), ***(p < 0.005) indicate significant differences.
Figure 3: Phase contrast and LIVE DEAD fluorescence micrographs showing cellular morphology and live and dead A549 cells in (a) and (e) control untreated, 24 h post treatment with (b) and f) 80 yM, (c) and (g) 160 yM and (d) and (h) 320 yM concentrations of Berberine. (200x Magnification). Green Calcein fluorescence represents Live cells and red EthD-1-stained nucleus represents dead cells.
The morphological changes in A549 cells after BBR induced chemotoxicity are shown in Fig 3. The control untreated cells (Fig. 3 (a) and (e)) showed healthy morphology with intact cell membrane. The number of cells in BBR treated group, showed gradual decrease in cell population at 80 |iM and 160 |iM (Fig. 3 (b) (c), (f) and (g)), with many cells showing rounded off morphology at the highest concentration of 320 |iM (Fig. 3 (d) and (h)).
As shown in Fig. 4 (a), treatment of A549 cells with various concentrations of BBR followed by light exposure at fixed light dose of 2 J/cm2 led to concentration dependent increase in cytotoxicity as compared to respective BBR unirradiated treatment groups. The IC50 and IC90 doses of ~ 11 |iM and 52 |iM is determined for irradiated groups (Fig. 4 (b)).
(a) (b)
100
Cone, of BBR (MM) Log Cone. Of BBR (MM)
Figure 4: (a) Concentration dependent (0, 2.5, 5, 10, 20, 40 and 63 yM) phototoxicity induced by Berberine (BBR) in A549 cells at a fixed light dose of 2 J/cm2 with 405 nm blue laser, determined by ATP proliferation assay (b) sigmoidal concentration-response curve of BBR treatment with and without light irradiation. Data points in the graphs are represented as mean ± standard errors from experiments repeated in
triplicates. *(p <0.05), **(p <0.01), ***(p < 0.005) indicate significant difference. In graph (a) statistical significance is determined between irradiated and unirradiated groups of each BBR concentration.
Unirradiated Irradiated (2J/cm2)
Figure 5: Representative phase contrast and LIVE DEAD fluorescence micrographs showing cellular morphology, live and dead A549 cells in control untreated and unirradiated (a) and (b), control untreated and irradiated (c) and (d), 24 h post treatment with 11 yM of Berberine (BBR) unirradiated (e) and (f) and irradiated (g) and (h), 63 yM of Berberine (BBR) unirradiated (i) and (j) and irradiated (k) and (l). (200x Magnification). Green Calcein fluorescence represents live cells and red EthD-1-stained nucleus represents
dead cells. Red arrows show rounded and damaged cells.
The changes in cellular morphology and viability observed with and without irradiated following BBR treatment is shown in Fig. 5. The irradiated group at BBR IC50 concentration (~11 pM) showed rounded up cellular morphology in certain cell population representing early signs of cell death (Fig. 5 (g) and (h)). While 63 |iM BBR concentration upon irradiation showed significant morphological changes with cells showing rounding up, detachment from culture plates, membrane damage and strong red fluorescent nuclei representing cell death (Fig. 5 (k) and (l)).
In the present study, ATP assay results demonstrate that the combined chemo and phototoxicity with BBR treatment at 63 |iM concentration resulted in ~95% loss in cancer cell viability in comparison to the 320 |iM BBR unirradiated group which showed a loss of cell viability by only 75%. Even the IC50 dose in the irradiated group is significantly low (~11 pM) compared to the unirradiated group (63 pM). Further, the morphological and viability analysis (Fig 5) also showed significant cell damage and death in large cell population of irradiated groups in comparison to unirradiated group. Thus, our preliminary results suggested, the combination of chemotoxic potential of BBR with its induced phototoxicity instigates an effective cytotoxic effect against lung cancer cells at very low concentrations which cannot be achieved without PDT.
In summary, plant-based compound BBR is a potential candidate to augment the anticancer action in combined chemo and phototoxic treatment and thus offers the advantage of its application as natural product-based cancer prevention and therapy. However, further research is warranted to provide mechanistic insights at protein, gene, and transcriptome levels to identify the exact cell death signaling pathway induced by the combination therapy.
ACKNOWLEDGEMENTS
The authors sincerely thank the South African Research Chairs initiative of the Department of science and technology and National Research Foundation (NRF) of South Africa, South African Medical Research Council (SAMRC) and Laser Re-search Centre (LRC), University of Johannesburg.
Research reported in this review article was supported by the South African Medical Research Council (SAMRC) through its Division of Research Capacity Development under the Research Capacity Development Initiative from funding received from the South African National Treasury. The content and findings reported/ illustrated are the sole deduction, view and responsibility of the researcher and do not reflect the official position and sentiments of the SAMRC.
FUNDING
This work is supported by the South African Research Chairs initiative of the Department of science and technology and National Research Foundation of South Africa (Grant No 98337), South African Medical Research Council (Grant No. SAMRC EIP007/2021), NRF Research Development Grants for Y-Rated Researchers (Grant No: 137788) as well as grants received from the University Research Committee (URC), University of Johannesburg and the Council for Scientific and Industrial Research (CSIR)-National Laser Centre (NLC).
CONFLICTS OF INTEREST
The authors declare no conflict of interest.
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