Photoactive metabolite mediated photodynamic therapy of Rhabdomyosarcoma cell lines using medicinal plants and Doxorubicin co-treatments

Abstract

Background: Medicinal plant-mediated combinational therapies have gained importance globally due to minimal side effects and enhanced treatment outcomes compared to single-drug modalities. We aimed to analyze the cytotoxic potential of each conventional treatment i.e., photodynamic therapy (PDT), chemotherapy (doxorubicin hydrochloride; Dox-HCl) with or without various concentrations of medicinal plant extracts (PE) on soft tissue cancer Rhabdomyosarcoma (RD) cell line.

Methods: The Rhabdomyosarcoma (RD) cell line was cultured and treated with Photosensitizer (Photosense (AlPc4)), Chemo (Dox-HCl), and their combinations with different concentrations of each plant extract i.e., Thuja occidentalis, Moringa oleifera, Solanum surattense. For the source of illumination, a Diode laser (λ = 630 nm ± 1 nm, P_max = 1.5 mW) was used. Photosensitizer uptake time (∼ 45 min) was optimized through spectrophotometric measurements (absorption spectroscopy). Drug response of each treatment arm was assessed post 24 h of administration using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5- 5-diphenyl-2 H- tetrazolium bromide (MTT) assay.

Results: PE-mediated Chemo-Photodynamic therapy (PDT) exhibited synergistic effects (CI < 1). Moreover, Rhabdomyosarcoma culture pretreated with various plant extracts for 24 h exhibited significant inhibition of cell viability however most effective outcomes were shown by low and high doses of Moringa oleifera compared to other plant extracts. Post low doses treated culture with all plant extracts followed by PDT came up with more effectiveness when compared to all di-therapy treatments.

Conclusion: The general outcome of this work shows that the ethanolic plant extracts (higher doses) promote the death of cancerous cells in a dose-dependent way and combining Dox-HCl and photo-mediated photodynamic therapy can yield better therapeutic outcomes.

Keywords: Absorption spectroscopy; Combination index; Medicinal plants; Photodynamic therapy; Photosensitizer; Rhabdomyosarcoma cancer.

Fig. 1

Photodynamic therapy setup

Fig. 2

Optimal time for maximum uptake of Photosense in RD cell culture

Fig. 3

Individual cell viability of lower doses of different treatment arms (Moringa oleifera; MO, Thuja occidentalis; Thuja, and Solanum surattense; Sn at 50 µg/ml); chemo drug (Dox-HCl) and photosensitizer phthalocyanine (µM)) on Rhbodmayosarcoma cell culture. Data indicated the results of three independent experiments (n = 3). Asterisks * and *** show significant difference at p < 0.05 and p < 0.001 versus the control group. Data was analyzed by one-way ANOVA followed by Tukey’s multiple comparison test

Fig. 4

Cytotoxicity of Moringa oleifera, Thuja occidentalis, and Solanum surattense at 150 µg/ml concentration. Chemo drug and phthalocyanine (Photosense) were administered at 150 µM doses. Data indicated the results of three independent experiments (n=3). Asterisks *** and **** show significant difference at p<0.001 and p<0.0001 versus the control group, whereas + and ++ showed significant difference at p<0.05 and p<0.01 versus the chemo 150 group. ### and #### indicated significant difference at p<0.001 and p<0.0001 versus PS 150 treatment group. × showed a comparison at p<0.05 between different plant extracts at high doses. Data was analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. Mo: Moringa oleifera, Thuj: Thuja occidentalis, Sn: Solanum surattense, Ps: Photosense, Chemo: Doxorubicin chemotherapy

Fig. 5

Effect of Di-combination of higher concentration (150 µg/ml) of Plant extracts, chemo (Dox-HCl), and photosensitizer on cell proliferation of Rhabdomyosarcoma cells. PS incubated for 45 min post 24 h of Plant & chemo drug. Data indicated the results of three independent experiments (n = 3). Asterisks **, ***, and **** shows significant differences at p < 0.01, p < 0.001, and p < 0.0001 versus the control group, whereas + shows a significant difference of Mo 150 + Chemo150 group at p < 0.05 versus Thuj150 + Chemo 150 group. # indicated significant difference of Sn 150 + Chemo 150 group at p < 0.05 versus Sn 150 + PS 50 group. Non-significant difference was noticed between Mo150andChemo150 versus Mo150 + Ps50 and Thuj150andChemo150 versus Thuj150 + Ps50 groups. Data was analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. Mo: Moringa oleifera, Thuj: Thuja occidentalis, Sn: Solanum surattense, Ps: Photosense. Chemo: Doxorubicin chemotherapy

Fig. 6

Tri-combination with a low dose of PS (50 µM), a higher dose of Chemo (150µM), and different doses of plants (50,150 µg/ml) followed by Photosense^®-mediated PDT. Data indicated the results of three independent experiments (n = 3). Asterisks **** show a significant difference at p < 0.0001 versus the control group. ++++ shows significant difference at p < 0.0001 of Mo 50 + Ps50 + Chemo150 treatment group versus Mo150 + PS 50 + Chemo150 treatment groups. ××× indicated significant difference at p < 0.001 of Thuj50 + Ps50 + Chemo150 versus Thuj 150 + PS 50 + Chemo150 treatment groups. ## indicated significant difference at p < 0.01 of Mo150 + Ps 50 + Chemo 150 treatment group versus Thuj150 + PS 50 + Chemo150 and Sn150 + Ps50 + Chemo150 treatment groups. Ø indicated a significant difference at p < 0.05 of Sn50 + Ps50 + Chemo150 versus Sn150 + PS 50 + Chemo150 treatment groups. Data was analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. Mo: Moringa oleifera, Thuj: Thuja occidentalis, Sn: Solanum surattense, Ps: Photosense, Chemo: Doxorubicin

Fig. 7

Plant extracts (Moringa oleifera, Thuja occidentalis, and Solanum surattense) 150 µg/ml chemo 50 µM, (Dox-HCl) and photosensitizer150 µM, individual cytotoxicity on rhabdomyosarcoma cell culture. Data indicated the results of three independent experiments (n = 3). Asterisks **** showed a significant difference at p < 0.0001 versus the control group. + showed significant difference at p < 0.05 of Mo150 + PS150 + Chemo50 group versus Sn150 + PS150 + Chemo50 groups

Fig. 8

Morphological changes (% cell survival) of various treatment groups in the presence of light. (a) Control (100%), (b)Moringa oleifera (MO, 28%), (c)Solanum surattense (Sn, 37%) (d)Thuja occidentalis (Thuj, 46%). PS: photosense, Chemo: Doxorubicin chemotherapeutic