Hypericin-mediated Photodynamic Therapy Research Articles

Hypericin-mediated photodynamic therapy inhibits metastasis and EMT of colorectal cancer cells by regulating RhoA-ROCK1 signaling pathway.

Colorectal cancer (CRC) is significantly contributed to global cancer mortality rates. Treating CRC is particularly challenging due to metastasis and drug resistance. There is a pressing need for new treatment strategies against metastatic CRC. Photodynamic therapy (PDT) offers a well-established, minimally invasive treatment option for cancer with limited side effects. Hypericin (HYP), a potent photosensitizer for PDT, has been documented to induce cytotoxicity and apoptosis in various types of cancers. However, there are few reports on the inhibitory effects of HYP-mediated PDT on the metastatic ability of CRC cells. Here, we evaluate the inhibitory effects of HYP-mediated PDT against metastatic CRC cells and define its underlying mechanisms. Wound-healing and Transwell assays show that HYP-mediated PDT suppresses migration and invasion of CRC cells. F-actin visualization assays indicate HYP-mediated PDT decreases F-actin formation in CRC cells. TEM assays reveal HYP-mediated PDT disrupts pseudopodia formation of CRC cells. Mechanistically, immunofluorescence and western blotting results show that HYP-mediated PDT upregulates E-cadherin and downregulates N-cadherin and Vimentin. HYP-mediated PDT also suppresses key EMT regulators, including Snail, MMP9, ZEB1 and α-SMA. Additionally, the expressions of RhoA and ROCK1 are downregulated by HYP-mediated PDT. Together, these findings suggest that HYP-mediated PDT inhibits the migration and invasion of HCT116 and SW620 cells by modulating EMT and RhoA-ROCK1 signaling pathway. Thus, HYP-mediated PDT presents a potential therapeutic option for CRC.

The contradictory role of febuxostat in ABCG2 expression and potentiating hypericin-mediated photodynamic therapy in colorectal cancers.

Photodynamic Therapy (PDT) is an emerging method to treat colorectal cancers (CRC). Hypericin (HYP) is an effective mediator of PDT and the ABCG2 inhibitor, Febuxostat (FBX) could augment PDT. HT29 and HEK293 cells showed light dependant cytotoxic response to PDT in both 2D and 3D cell models. FBX co-treatment was not found to improve PDT cytotoxicity. Next, ABCG2 protein expression was observed in HT29 but not in HEK293 cells. However, ABCG2 gene expression analysis did not support protein expression results as ABCG2 gene expression results were found to be higher in HEK293 cells. Although HYP treatment was found to significantly reduce ABCG2 gene expression levels in both cell lines, FBX treatment partially restored ABCG2 gene expression. Our findings indicate that FBX co-treatment may not be suitable for augmenting HYP-mediated PDT in CRC but could potentially be useful for other applications.

Effect of hypericin-mediated photodynamic therapy on the secretion of selected TNF superfamily receptors by oral cancer cells

Effect of hypericin-mediated photodynamic therapy on the secretion of selected TNF superfamily receptors by oral cancer cells

Immunomodulatory Effect of Hypericin-Mediated Photodynamic Therapy on Oral Cancer Cells.

In 2020, there were 377,713 new oral and lip cancer diagnoses and 177,757 deaths. Oral cancer is a malignancy of the head and neck region, and 90% of cases are squamous cell carcinomas (OSCCs). One of the alternative methods of treating pre-cancerous lesions and oral cancer is photodynamic therapy (PDT). In addition to the cytotoxic effect, an important mechanism of PDT action is the immunomodulatory effect. This study used the OSCC (SCC-25) cell line and the healthy gingival fibroblast (HGF-1) line. A compound of natural origin-hypericin (HY)-was used as the photosensitizer (PS). The HY concentrations of 0-1 µM were used. After two hours of incubation with PS, the cells were irradiated with light doses of 0-20 J/cm2. The MTT test determined sublethal doses of PDT. Cell supernatants subjected to sublethal PDT were assessed for interleukin 6 (IL-6), soluble IL-6 receptor alpha (sIL-6Ralfa), sIL-6Rbeta, IL-8, IL-10, IL-11 IL-20, IL-32, and Pentraxin-3 using the Bio-Plex ProTM Assay. The phototoxic effect was observed starting with a light dose of 5 J/cm2 and amplified with increasing HY concentration and a light dose. HY-PDT affected the SCC-25 cell secretion of sIL-6Rbeta, IL-20, and Pentraxin-3. HY alone increased IL-8 secretion. In the case of HGF-1, the effect of HY-PDT on the secretion of IL-8 and IL-32 was found.

Effects of hypericin-mediated photodynamic therapy on GM-CSF, MIF, VCAM-1 and ICAM-1 secretion in colorectal cancer cells in vitro

Introduction and aim. Photodynamic therapy with hypericin (HYP-PDT) is gaining importance as a potential treatment method for malignant neoplasms. The following study investigated whether HYP-PDT influences the secretion of certain factors of colon cancer cells in vitro. Material and methods. Two colon cancer cell lines were used in this experiment: SW480 and SW620. The cells were properly prepared and then treated with photodynamic therapy with hypericin at concentrations of 0.25 μM and 0.5 μM and irradiation at the doses of 1 J/cm 2 , 5 J/cm 2 and 10 J/cm 2 . After using HYP-PDT, changes in the concentrations of four factors: GM-CSF, MIF, VCAM-1 and ICAM-1 were analyzed in the test tubes. Results. In the case of SW480 cells: a notable decrease in GM-CSF, MIF, VCAM-1 and ICAM-1 secretion was noted after HYP-PDT. In the case of SW620 cells, after HYP-PDT: no effect on GM-CSF secretion was noted; it inhibited the secretion of VCAM-1 and MIF and increased the secretion of ICAM-1. Conclusion. Photodynamic therapy with hypericin shows immunomodulatory potential in an in vitro cell line experiments. This may indicate its possible ability to modify the course of malignant tumors and therefore requires further research.

Effect of Hypericin-Mediated Photodynamic Therapy on the Secretion of Soluble TNF Receptors by Oral Cancer Cells.

Squamous cell carcinoma is the most common cancer of the head and neck region. In addition to the classic surgical treatment method, alternative therapy methods are sought. One such method is photodynamic therapy (PDT). In addition to the direct cytotoxic effect, it is essential to determine the effect of PDT on persistent tumor cells. The study used the SCC-25 oral squamous cell carcinoma (OSCC) cell line and the HGF-1 healthy gingival fibroblast line. A compound of natural origin-hypericin (HY)-was used as a photosensitizer (PS) at concentrations of 0-1 µM. After two hours of incubation with the PS, the cells were irradiated with light doses of 0-20 J/cm2. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test was used to determine sublethal doses of PDT. Cell supernatants subjected to sublethal PDT were assessed for soluble tumor necrosis alpha receptors (sTNF-R1, sTNF-R2). The phototoxic effect was observed starting with a light dose of 5 J/cm2 and amplified with the increase in HY concentration and light dose. A statistically significant increase in sTNF-R1 secretion by SCC-25 cells was demonstrated after the PDT with 0.5 µM HY and irradiation with 2 J/cm2 (sTNF-R1 concentration = 189.19 pg/mL ± 2.60) compared to the control without HY and irradiated with the same dose of light (sTNF-R1 concentration = 108.94 pg/mL ± 0.99). The baseline production of sTNF-R1 was lower for HGF-1 than for SCC-25, and secretion was not affected by the PDT. The PDT had no effect on the sTNF-R2 production in the SCC-25 or HGF-1 lines.

Promising onychomycosis treatment with hypericin-mediated photodynamic therapy: Case reports

BackgroundOnychomycosis (OM) is a common nail plate disorder caused by dermatophyte molds, yeasts, and non-dermatophyte molds, which use keratin in the nail plate as an energy source. OM is characterized by dyschromia, increased nail thickness, subungual hyperkeratosis, and onychodystrophy, and is typically treated with conventional antifungals despite frequent reports of toxicity, fungal resistance, and OM recurrence. Photodynamic therapy (PDT) with hypericin (Hyp) as a photosensitizer (PS) stands out as a promising therapeutic modality. When excited by a specific wavelength of light and in the presence of oxygen, to lead to photochemical and photobiological reactions on the selected targets. MethodsOM diagnosis was made in three suspected cases, and the causative agents were identified by classical and molecular methods, and confirmed by attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). Susceptibility of planktonic cells of the clinical isolates to conventional antifungals and PDT-Hyp was evaluated, and photoacoustic spectroscopy (PAS) of Hyp permeation in nail fragments ex vivo was analyzed. Furthermore, the patients opted to undergo PDT-Hyp treatment and were subsequently followed up. The protocol was approved by the human ethics committee (CAAE, number 14107419.4.0000.0104). ResultsThe etiological agents of OM in patients ID 01 and ID 02 belonged to the Fusarium solani species complex, being identified as Fusarium keratoplasticum (CMRP 5514) and Fusarium solani (CMRP 5515), respectively. For patient ID 03, the OM agent was identified as Trichophyton rubrum (CMRP 5516). PDT-Hyp demonstrated a fungicidal effect in vitro, with reductions of p3 log10 (p < 0.0051 and p < 0.0001), and the PAS analyses indicated that Hyp could completely permeate through both healthy and OM-affected nails. After four sessions of PDT-Hyp, mycological cure was observed in all three cases, and after seven months, clinical cure was confirmed. ConclusionPDT-Hyp showed satisfactory results in terms of efficacy and safety, and thus can be considered a promising therapy for the clinical treatment of OM.

Photobiomodulation and photodynamic therapy-induced switching of autophagy and apoptosis in human dermal fibroblasts.

Nowadays, photobiomodulation (PBM) in combination with chemotherapy or other therapeutic approaches is an attractive adjuvant modality for cancer treatment. Targeted destruction of cancer cells is one of the main advantages of photodynamic therapy (PDT). We have shown in previous studies that the combination of PBM at 808nm and hypericin-mediated PDT increases PDT efficacy in human glioblastoma cells U87 MG. The study presented here shows significant differences between U87 MG and non-cancerous human dermal fibroblasts (HDF) cells treated by PBM and PDT. This study focuses on mitochondria because PBM mainly affects these organelles. We demonstrated that an interplay between mitochondrial and autophagic proteins plays a crucial role in the response of HDF cells to PBM and PDT. Fluorescence microscopy, flow cytometry, and Western blot analysis were used to examine the autophagic profile of HDF cells after these treatments. An increase in ubiquitin, SQSTM1, LC3BII, and cytochrome c was accompanied by a decrease in M6PR, ATG16L1, and Opa1 in HDF cells exposed to PBM and PDT. Overall, we observed that the switching of autophagy and apoptosis is dose-dependent and also occurs independently of PBM in HDF cells after hypericin-mediated PDT. However, PBM might preferentially induce autophagy in noncancer cells, which might escape apoptosis under certain conditions.

Autophagy and Apoptosis Induced in U87 MG Glioblastoma Cells by Hypericin-Mediated Photodynamic Therapy Can Be Photobiomodulated with 808 nm Light

Glioblastoma is one of the most aggressive types of tumors. Although few treatment options are currently available, new modalities are needed to improve prognosis. In this context, photodynamic therapy (PDT) is a promising adjuvant treatment modality. In the present work, hypericin-mediated PDT (hypericin-PDT, 2 J/cm2) of U87 MG cells is combined with (2 min, 15 mW/cm2 at 808 nm) photobiomodulation (PBM). We observed that PBM stimulates autophagy, which, in combination with PDT, increases the treatment efficacy and leads to apoptosis. Confocal fluorescence microscopy, cytotoxicity assays and Western blot were used to monitor apoptotic and autophagic processes in these cells. Destabilization of lysosomes, mitochondria and the Golgi apparatus led to an increase in lactate dehydrogenase activity, oxidative stress levels, LC3-II, and caspase-3, as well as a decrease of the PKCα and STAT3 protein levels in response to hypericin-PDT subcellular concentration in U87 MG cells. Our results indicate that therapeutic hypericin concentrations can be reduced when PDT is combined with PBM. This will likely allow to reduce the damage induced in surrounding healthy tissues when PBM-hypericin-PDT is used for in vivo tumor treatments.

Near-infrared Transillumination and Photodynamic Therapy Using Hypericin in Animal Laryngeal Tumors.

We aimed to validate a pilot study of photodiagnosis using near infrared (NIR) transillumination and assess the clinical efficacy of hypericin-mediated photodynamic therapy (HYP-PDT) in a rabbit laryngeal cancer model in order to develop a novel therapeutic modality with complete remission and preservation of the functional organ. (1) In vitro study: VX tumor cells were subcultured and subjected to HYP-PDT. (2) In vivo study: A laryngeal cancer model was developed in which 12 rabbits were inoculated with a VX tumor suspension in the submucosal area of the left vocal fold using a transoral approach. All rabbits underwent NIR transillumination using light with a wavelength of 780nm. The survival periods of the three treatment groups (6 rabbits in Group A: HYP-PDT, 3 each in Groups B and C: laser irradiation or HYP administration only) were analyzed. The higher the HYP concentration, the lower the VX cell viability in response to HYP-PDT using 590nm LED. Following HYP-PDT, small tumors in Group A-1 rabbits healed completely and the animals demonstrated a long survival period, and larger tumors in Group A-2 healed partially with a survival period that extended over 3weeks after inoculation. The survival of Groups B and C were not different over the first 3weeks of the study, and were shorter than in Group A. We found HYP-PDT could be a curative therapy for early-stage cancers that may also preserve organ function, and may inhibit tumor progression and metastasis during advanced stages of laryngeal cancer.

Hypericin-mediated photodynamic therapy enhances gemcitabine induced Capan-2 cell apoptosis via inhibiting NADPH level.

The combination of gemcitabine (Gem) and hypericin (HY) enhances the apoptosis of Capan-2 cells, providing a promising option for the treatment of pancreatic cancer. Our study further explored the cytotoxic mechanism of HY combined with chemotherapy drugs on pancreatic cancer. The proliferation rate of the cells assayed with the MTT method. The ROS (reactive oxygen species) levels of each treatment were evaluated by DCFH-DA oxidisation methods. The activity of glutathione reductase and the levels of reduced glutathione (GSH) and oxidised glutathione (GSSG) were assessed using assay kits. The expression levels of apoptosis-related proteins were analysed by western blotting. The activity of glucose-6-phosphate dehydrogenase (G6PDH), a key enzyme of the pentose phosphate pathway, significantly decreased in Gem + HY groups, however, the ROS level enhanced accompanying with GSH depleting, mitochondrial membrane depolarisation and cytochrome C release. Gem + HY inhibits the expression of Bcl-2 but stimulates Bax level, triggering caspase activation and PARP cleavage and thus promoted apoptosis of Capan-2 cells. We demonstrated that Gem combined HY-PDT could inhibit the proliferation of Capan-2 cells and induce cell apoptosis. HY-PDT combined with Gem had a great potential on pancreatic cancer treatment clinically.

Hypericin-mediated photodynamic therapy inhibits growth of colorectal cancer cells via inducing S phase cell cycle arrest and apoptosis

Colorectal cancer (CRC) is one type of cancer with high morbidity and mortality worldwide. Photodynamic therapy (PDT), a promising new therapeutic approach for cancer, induces tumor damage through photosensitizer-mediated oxidative cytotoxicity. Hypericin is a powerful photosensitizer with pronounced tumor-localizing properties. In this study, we investigated the phototoxic effects of hypericin-mediated PDT (HYP-PDT) in HCT116 and SW620 cells. We validated that HYP-PDT inhibited cell proliferation, triggered intracellular reactive oxygen species generation, induced S phase cell cycle arrest and apoptosis of HCT116 and SW620 cells. Mechanistically, the results of western blot showed that HYP-PDT downregulated CDK2 expression through decreasing the CDC25A protein, which resulted in the decrease of CDK2/Cyclin A complex. Additionally, HYP-PDT induced DNA damage as evidenced by ATM activation and upregulation of p-H2AX. Further investigation showed that HYP-PDT significantly increased Bax expression and decreased Bcl-2 expression, and then, upregulated the expression of cleaved caspase-9, cleaved caspase-3 and cleaved PARP, thereby inducing apoptosis in HCT116 and SW620 cells. In conclusion, our results indicated that the CDC25A/CDK2/Cyclin A pathway and the mitochondrial apoptosis pathway were involved in HYP-PDT induced S phase cell cycle arrest and apoptosis in colorectal cancer cells, which shows HYP could be a probable candidate used for treating colorectal cancer.

Hypericin-mediated photodynamic therapy for the treatment of cancer: a review.

Hypericin is a polycyclic aromatic naphthodianthrone that occurs naturally. It is also an active ingredient in some species of the genus Hypericum. Emerging evidence suggests that hypericin has attracted great attention as a potential anticancer drug and exhibits remarkable antiproliferative effect upon irradiation on various tumour cells. This paper aims to summarise the anticancer effect and molecular mechanisms modulated by hypericin-medicated photodynamic therapy and its potential role in the cancer treatment. Hypericin-medicated photodynamic therapy could inhibit the proliferation of various tumour cells including bladder, colon, breast, cervical, glioma, leukaemia, hepatic, melanoma, lymphoma and lung cancers. The effect is primarily mediated by p38 mitogen-activated protein kinase (MAPK), JNK, PI3K, CCAAT-enhancer-binding protein homologous protein (CHOP)/TRIB3/Akt/mTOR, TRAIL/TRAIL-receptor, c-Met and Ephrin-Eph, the mitochondria and extrinsic signalling pathways. Furthermore, hypericin-medicated photodynamic therapy in conjunction with chemotherapeutic agents or targeted therapies is more effective in inhibiting the growth of tumour cells. During the past few decades, the anticancer properties of photoactivated hypericin have been extensively investigated. Hypericin-medicated photodynamic therapy can modulate a variety of proteins and genes and exhibit a great potential to be used as a therapeutic agent for various types of cancer.

Abstract B35: Studies on photo-sensitivity of a glycol porphyrin derivative and its anti-tumor efficacy

Abstract Photodynamic therapy (PDT) has attracted great attention in cancer treatment. Porphyrin derivative with ethylene glycol chains linked to meta position of meso-tetraphenylporphyrin, mTPP(EG)4, is one of assumed compounds that can be used for treatment of cancer in photodynamic therapy (PDT). The aim of the study is to demonstrate the photodynamic efficacy in vitro and in vivo as well as to analyze its therapeutic mechanism. The cell viability, cell surface exposure of immunogenic molecules, maturation and phagocytic capacity of PDT-treated tumor cells were monitored by flow cytometry. The kinetics of key components of ER stress-mediated apoptotic pathway was analyzed by Western blotting. Our first results indicate a new and attractive possibility of immunogenic cell death induction. Initiation of immunogenic cell death by PDT was successfully demonstrated in vitro on human and mouse tumor cell lines. The effects of mTPP(EG)4 were compared with the effects of mTPP(EG)4 without subsequent photoactivation, hypericin-mediated PDT, and high hydrostatic pressure. In vivo assays evaluated immunogenicity of mTPP(EG)4 in mice and assessed the ability of PDT treated cells to induce effective immunity against transplanted syngeneic tumors (mouse TRAMP-C2, TC-1 and B16). The results obtained in this study will provide a basis for possible further preclinical experiments with mTPP(EG)4 or other derivatives, testing their potential in PDT and development of new antitumor therapies. Citation Format: Sarka Vosahlikova, Irena Kusova Moserova, Jarmila Kralova, Milan Reinis, Romana Mikyskova, Jitka Palich Fucikova. Studies on photo-sensitivity of a glycol porphyrin derivative and its anti-tumor efficacy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B35.

The Influence of Hypericin-Mediated Photodynamic Therapy on Interleukin-8 and -10 Secretion in Colon Cancer Cells.

The aim of this study was to measure the secretion of interleukin (IL)-8 and -10 during an elicited immune response following sublethal doses of hypericin-mediated photodynamic therapy (HY-PDT) in experimental models of residual colon cancer cells in vitro. Investigations were performed on the cancer cell lines SW480 and SW620. Each cell line was exposed to 3 different concentrations of the photosensitizer HY and various doses of irradiation. The cell metabolic activity using an MTT assay was performed and then the measurement of IL-8 and IL-10 secretion was achieved using the Bio-Plex ProTMAssay. There was a statistically significant amplification of IL-8 secretion during HY-PDT in the SW620 cell line (at 1 J/cm2: P = .01, 5 J/cm2: P = .002, and 10 J/cm2: P = .025) and a statistically significant decrease in IL-8 during HY-PDT in the SW480 cell line (at 1 J/cm2: P = .05, 5 J/cm2: P = .035, and 10 J/cm2: P = .035). No statistically significant differences in IL-10 concentration were found following HY-PDT in the SW480 (at 1 J/cm2: P > .4, 5 J/cm2: P = .1, and 10 J/cm2: P = .075) or in the SW620 cell line (at 1 J/cm2: P > .4, 5 J/cm2: P > .4, and 10 J/cm2: P > .4). HY-PDT can both eliminate and control a primary tumor via cytotoxic effects, and at sublethal doses, it can affect IL release by colon cancer cells. In this experiment, this influence depended on the level of tumor cell metastatic activity.

An Electron paramagnetic resonance (EPR) spin labeling study in HT-29 Colon adenocarcinoma cells after Hypericin-mediated photodynamic therapy

BackgroundColon cancer affects 1.23 million people worldwide and is the third most common malignant disease in men and the second in women. The only curative treatment is surgical resection, but a significant number of patients develop local recurrence or distant metastases. One of the alternative treatment methods for colon cancer is photodynamic therapy (PDT). In recent years, hypericin (HYP) derived from Hypericum perforatum has been suggested as a strong candidate photosensitizer for PDT. Our interest is focused on the biophysical changes in colon cancer cells in relation to HYP-mediated PDT.ResultsIn this study, HYP-mediated PDT at 0.04, 0.08 or 0.15 μM HYP concentrations was performed in HT-29 colon adenocarcinoma cells and the Electron Paramagnetic Resonance (EPR) spectra of the spin labeled cells were obtained. Plasma membranes are already heterogeneous structures; the presence of cancer cells increased the heterogeneity and also fluidity of the plasma membranes. Therefore, the obtained spectra were evaluated by EPRSIMC program, which provides the calculation of heterogeneous structures up to four spectral components with different fluidity characteristics. Generally, two motional patterns were obtained from calculations and the number of them increased at the highest concentration. As the order parameters of the most populated components compared, an increase was observed depending on the HYP concentration. However, because of the heterogeneous structure of membrane, the order parameters of the less populated components did not exhibit a regular distribution.ConclusionAfter HYP-mediated PDT, concentration dependent changes were observed in the domain parameters indicating an increase in the HYP accumulation.

Breast cancer resistance protein is the enemy of hypericin accumulation and toxicity of hypericin-mediated photodynamic therapy

Breast cancer resistance protein (BCRP) belongs to the family of ATP-binding cassette (ABC) transporters, overexpression of which can confer a multidrug-resistant phenotype in cancer cells and tumors. BCRP mediates efflux of numerous xenobiotics, including various chemotherapeutic agents and photosensitizers. Hypericin (HY) is a naturally-occurring photosensitizer synthesized by plants of the genus Hypericum. Our recently published results indicate that accumulation of HY in cancer cells of different tissue origin can be affected mostly by BCRP. Considering all known facts, the main goal of this study was to verify whether not only HY accumulation but also toxicity of HY-mediated photodynamic therapy (PDT) can be affected by the presence of some ABC transporters. To specifically prove our hypothesis, we used an experimental model of human leukemia cell lines differing in the expression level of the main drug efflux transporters P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and BCRP. The lowest HY accumulation, and consequently the highest resistance to HY-PDT, was found in cells overexpressing BCRP. Moreover, pretreatment with BCRP inhibitor Ko143 significantly increased HY accumulation and sensitized cells to HY-PDT. Therefore, our findings represent direct evidence that BCRP is the nemesis of HY accumulation and toxicity of HY-PDT. Thus, we should emphasize that individualized screening for BCRP expression and activity may represent a useful tool for prediction of HY-mediated photodynamic diagnosis (PDD) or PDT effectiveness.

Inhibiting ABCG2 could potentially enhance the efficacy of hypericin-mediated photodynamic therapy in spheroidal cell models of colorectal cancer.

Photodynamic Therapy (PDT) is an attractive modality for treating solid cancers. This study evaluates the efficacy of Hypericin-PDT as a cytotoxic therapy in colorectal cancer (CRC), using 2D cell cultures and 3D multicellular tumour spheroids. Spheroids were generated through forced-floating and agitation-based techniques. 2D and spheroid models of HT29 and HCT116 CRC cells were incubated with Hypericin (0-200 nM) for 16 h. Cultures were irradiated with light (1 J/cm2) and cytotoxicity assessed using Propidium Iodide fluorescence. Expression of ABCG2 protein was assessed by immunoassays in 2D and spheroid cultures. The effect of ABCG2 inhibition, using 10 μM Ko143, on cytotoxicity following Hypericin-PDT was evaluated. Hypericin-PDT produced a significant reduction in HT29 (p < 0.0001) and HCT116 (p < 0.0001) cell viability in 2D cultures, with negligible non-phototoxicity. Spheroids were more resistant than 2D cultures to Hypericin-PDT (HT29: p = 0.003, HCT116: p = 0.006) and had a greater expression of ABCG2. Inhibition of ABCG2 in spheroids with Ko143 resulted in an enhanced Hypericin-PDT effect compared to Hypericin-PDT alone (HT29: p = 0.04, HCT116: p = 0.01). Hypericin-PDT has reduced efficacy in CRC spheroids as compared to 2D cultures, which may be attributable through upregulation in ABCG2. The clinical efficacy of Hypericin-PDT may be enhanced by ABCG2 inhibition.

Endoplasmic reticulum-targeting photosensitizer Hypericin confers chemo-sensitization towards oxaliplatin through inducing pro-death autophagy

Hypericin is an endoplasmic reticulum (ER)-located photosensitizer, which causes oxidative damage to ER during photodynamic therapy (PDT). Hypericin-mediated PDT (HY-PDT) has been confirmed to enhance chemo-sensitivity of oxaliplatin (L-OHP) in colon cancer cells. The present study reveals that autophagy plays a key role in chemosensitization during HY-PDT. We proved pro-death autophagy was required for sensitization and HY-PDT/L-OHP antitumor synergism. High dosage of HY-PDT induced autophagic cell death; while low dose of HY-PDT predominantly triggered protective autophagy and promoted cell proliferation. Low dose of HY-PDT reduced the cytotoxicity of L-OHP in oxaliplatin-resistant colon cancer cells. Different level of autophagy therefore contributed to the opposite effect of HY-PDT on cell fate and chemo-sensitivity. Furthermore, we revealed the role of CHOP as a regulator connecting pro-survival and pro-death autophagy under ER damage. High dose of HY-PDT induced massive ROS generation and severe ER stress, which then led to induction of CHOP. CHOP thereby activated CHOP/TRIB3/Akt/mTOR cascade and triggered autophagic cell death. Additionally, when apoptotic pathway was blocked, cells treated with high dose of HY-PDT preferentially underwent death through autophagic pathway. On the other hand, suppression of autophagy made cells more vulnerable to apoptosis under low dose of HY-PDT. These results provided new evidences for the clinical application of ER-targeting PDT in modifying chemosensitivity of colorectal cancer therapy.

YM155, a small molecule inhibitor of survivin expression, sensitizes cancer cells to hypericin-mediated photodynamic therapy.

Photodynamic therapy (PDT) represents a rapidly developing alternative treatment for various types of cancers. Although considered highly effective, cancer cells can exploit various mechanisms, including the upregulation of apoptosis inhibitors, to overcome the cytotoxic effect of PDT. Survivin, a member of the inhibitor of apoptosis protein family, is known to play a critical role in cancer progression and therapeutic resistance and therefore represents a potential therapeutic target. The aim of this study was to investigate whether YM155, a small molecule inhibitor of survivin expression, can potentiate the cytotoxic effect of hypericin-mediated PDT (HY-PDT). Accordingly, two cell lines resistant to HY-PDT, HT-29 (colorectal adenocarcinoma) and A549 (lung adenocarcinoma), were treated either with HY-PDT alone or in combination with YM155. The efficacy of different treatment regimens was assessed by MTT assay, flow cytometry analysis of metabolic activity, viability, phosphatidylserine externalisation, mitochondrial membrane potential and caspase-3 activity and immunoblotting for the cleavage of poly (ADP-ribose) polymerase (PARP). Here we show for the first time that the repression of survivin expression by YM155 is effective in sensitizing HT-29 and A549 cells to HY-PDT, as measured by the decrease in cell viability and induction of apoptosis. Combined treatment with hypericin and YM155 led to a more severe dissipation of the mitochondrial membrane potential and caused an increase in caspase-3 activation and subsequent PARP cleavage. Our results demonstrate that the repression of survivin expression by YM155 potentially represents a novel alternative strategy to increase the efficacy of HY-PDT in cancer cells that are otherwise weakly responsive or non-responsive to treatment.