Raktim Biswas

Modulation of EGFR and ROS induced cytochrome c release by combination of photodynamic therapy and carboplatin in human cultured head and neck cancer cells and tumor xenograft in nude mice.

Photodynamic therapy in combination with different treatment modalities has been evaluated to study the mechanism of cellular cytotoxicity and apoptosis in various forms of cancer. In the present study, human head and neck cancer cells were treated with radachlorin mediated photodynamic therapy and the chemotherapy drug, carboplatin singly or in combination. Several parameters were studied to check the enhanced cytotoxicity of combination therapy at different time interval. From the cell viability study by MTT assay, a 22% decrease in cell viability was observed in combination treatment. This enhanced activity of combination treatment was confirmed by cell migration assay and Hoechst PI staining. Generation of reactive oxygen species was observed and found to be higher than that of individual treatments. Cytochrome c was found to be released from mitochondria that also induced the enhance efficacy in combination treatment. The expression of other proteins like EGFR and PARP was also modulated with the time of incubation after treatment. In the tumor xenograft study in nude mouse model, the carboplatin treated group did not show any noticeable changes in tumor volume whereas tumor volume was reduced in PDT and the combination group. Though the difference in the reduction of the tumor size was not significant between PDT and combination group, there was a difference in the expression of EGFR between these two groups. Histologic study of the inhibition in tumor growth was also performed. Therefore, this study may provide an avenue of combating head and neck cancer by a combination of conventional chemotherapy and PDT.

Sulforaphene promotes Bax/Bcl2, MAPK-dependent human gastric cancer AGS cells apoptosis and inhibits migration via EGFR, p-ERK1/2 down-regulation.

Gastric cancer migration and invasion considered as main causes of this cancer-related death around the world. Sulforaphene (4-isothiocyanato-4R-(methylsulfinyl)-1-butene), a structural analog of sulforaphane, has been found to exhibit anticancer potential against different cancers. Our aim was to investigate whether dietary isothiocyanate sulforaphene (SFE) can promote human gastric cancer (AGS) cells apoptosis and inhibit migration. Cells were treated with various concentrations of SFE and cell viability, morphology, intracellular ROS, migration and different signaling protein expressions were investigated. The results indicate that SFE decreases AGS cell viability and induces apoptosis in a dose-dependent manner. Intracellular ROS generation, dose- and time-dependent Bax/Bcl2 alteration and signaling proteins like cytochrome c, Casp-3, Casp-8 and PARP-1 higher expression demonstrated the SFE-induced apoptotic pathway in AGS cells. Again, SFE induced apoptosis also accompanied by the phosphorylation of mitogen-activated protein kinases (MAPKs) like JNK and P-38. Moreover, dose-dependent EGFR, p-ERK1/2 down-regulation and cell migration inhibition at non-toxic concentration confirms SFE activity in AGS cell migration inhibition. Thus, this study demonstrated effective chemotherapeutic potential of SFE by inducing apoptisis as well as inhibiting migration and their preliminary mechanism for human gastric cancer management.

Combination with genistein enhances the efficacy of photodynamic therapy against human anaplastic thyroid cancer cells

Photodynamic therapy (PDT) has become one of the emerging options in management of cancer and other diseases. The major goal of PDT is to kill cancer cell without causing any adverse effect to the normal cells. PDT in combination of different therapeutic agents is being evaluated to improve the efficacy of treatment. Genistein, a soy ingredient, has widely been studied against different types of cancer. In the present study, combination of these two therapeutic methods has been studied to evaluate the enhanced effectiveness and find out the mechanism of action.

Chlorin e6 Derivative Radachlorin Mainly Accumulates in Mitochondria, Lysosome and Endoplasmic Reticulum and Shows High Affinity toward Tumors in Nude Mice in Photodynamic Therapy

The efficacy of photodynamic therapy (PDT) depends upon the amount of photosensitizer accumulated in the malignant tissues. Radachlorin is a popular photosensitizer used in photodynamic therapy to treat various types of cancer. In this study, we have studied the main organelles responsible for the accumulation of radachlorin in human anaplastic thyroid cancer in vitro and in vivo. The optimal time window for uptake and clearance of radachlorin also was studied. Confocal microscopic images confirmed that the radachlorin is mainly acquired by mitochondria and partially by lysosome and endoplasmic reticulum. Studies also showed that the maximum amount of radachlorin was accumulated within 3–6 h after the treatment. Radachlorin also showed a higher affinity toward malignant tumors compared to the other organs in mice xenograft model. Uptake of radachlorin reached an optimum amount within 6 h and most of the radachlorins were also cleared from the body in next 48 h. Therefore, detailed information regarding exact accumulation sites and a time window in which maximum amount of drug is accumulated and cleared were obtained by this study. Hence, not only the efficacy of the treatment can be increased but the phototoxicity after the treatment also can be controlled.

Carboplatin synergistically triggers the efficacy of photodynamic therapy via caspase 3-, 8-, and 12-dependent pathways in human anaplastic thyroid cancer cells

Anaplastic thyroid cancer is one of the most aggressive forms of malignancies which grow very rapidly. Several conventional methods have been applied for the treatment of anaplastic thyroid cancer, but most of them were not successful in complete recovery of the patients. Therefore, a combination of two or more conventional modalities is being applied nowadays for the treatment of this type of cancer. In this present study, the combination of photodynamic therapy (PDT) and chemotherapy has been studied in anaplastic thyroid cancer. Human anaplastic thyroid cancer cells FRO were treated with a chemotherapy drug, carboplatin (cis-diammine-1,1-cyclobutanedicarboxyl-ateplatinum II (CBDCA)), and radachlorin-mediated PDT individually and in combination. Several parameters like cytotoxicity assay by MTT, apoptosis study by annexin V and propidium iodide, cell cycle analysis by flow cytometry, confocal microscopic study, and Western blot analysis for different apoptosis-related proteins like Bax, cytochrome c, caspases 3, 9, 8, and 12, etc. were studied to check the efficacy of the combination treatment as well as to find out the mechanism of this enhanced efficacy. Results showed that both PDT and CBDCA can induce apoptosis in FRO cells. However, a synergistic efficacy was observed when the cells were treated with CBDCA and PDT in combination. Changes in mitochondrial membrane potential and an increase in reactive oxygen species generation were observed in combination treatments. The enhanced expression of different apoptotic pathway-related proteins like Bax, cytochrome c, caspase 3, caspase 8, caspase 12, etc. also confirmed the higher efficacy of combination treatment. Therefore, with this combination treatment, not only a higher efficacy can be achieved but also the effective dose of the chemotherapy drug can be reduced, and hence, the adverse side effects of the chemotherapy drugs can also be controlled.

Deregulation of EGFR/PI3K and activation of PTEN by photodynamic therapy combined with carboplatin in human anaplastic thyroid cancer cells and xenograft tumors in nude mice

Phosphatidylinositol-3-kinase (PI3K) is one of the major activated pathways involved in the progression of anaplastic thyroid cancer. The activated PI3K pathway starts from the overexpression of epidermal growth-factor receptor (EGFR) which plays a key role in cancer development and metastasis. However, a protein, PTEN negatively regulates the PI3K pathway. Here we studied the possibility of using a combination of conventional chemotherapy and photodynamic therapy to inhibit the growth of human anaplastic thyroid cancer in vitro and in vivo. Carboplatin (CBDCA) and radachlorin-photodynamic therapy (PDT) were used for the combination treatment of human anaplastic thyroid cancer cells FRO and tumor xenograft in athymic mice. Confocal microscopic and flow cytometric observations showed that cell death was mainly through an enhanced apoptosis with the combination of CBDCA and PDT. Generation of reactive oxygen species and dysfunction of mitochondrial membranes suggested that the enhanced apoptosis was achieved through the mitochondrial cell death pathway. This was confirmed by Western blot analysis of caspase 3, 9 expressions. Further analysis showed that the combination of CBDCA and PDT inhibited the expression of EGFR and PI3K with higher efficacy. PTEN also was activated more in this combination group. This suggests a combination of CBDCA and PDT modulates EGFR and PI3K as well as activates PTEN to inhibit tumor growth and induce apoptosis with an enhanced efficacy in anaplastic thyroid cancer.

Cisplatin enhances the efficacy of 5-aminolevulinic acid mediated photodynamic therapy in human head and neck squamous cell carcinoma.

Photodynamic therapy (PDT) has become a promising option for the treatment of head and neck, and other forms of cancer. 5-Aminolevulinic acid (ALA) is one of the popular photosensitizers used in PDT. It is a heme precursor and is converted to a photosensitizer protoporphyrin IX. In this present study, the combination of anticancer drug cisplatin (CDDP)- and ALA- mediated PDT was used to study the cytotoxicity in vitro as well as in vivo. Human head and neck cancer cells AMC-HN3 were treated with cisplatin- and ALA-mediated PDT individually, and also in combination. Several approaches like confocal microscopic study, cytotoxicity assay, etc have been performed to study the intracellular accumulation of protophorphyrin IX in cells and its effectiveness in PDT, when treated in combination with chemotherapy drug, cisplatin (CDDP). The combination of treatments efficacy was also studied in tumor xenograft model. Compared to the individual treatments, combination of CDDP and PDT was found to be more cytotoxic in AMC-HN3, and also more effective in reducing the tumor volume in mice xenograft. Thus, with the combined therapy, not only the efficacy of treatment can be enhanced, but the doses of the drugs can also be lowered. This in turn can reduce the side effects of the chemotherapy drugs. Therefore, this study may lead to a potential drug-PDT combination that may be a useful treatment modality for human head and neck cancer.

Synergistic effect of radachlorin mediated photodynamic therapy on propolis induced apoptosis in AMC-HN-4 cell lines via caspase dependent pathway.

BACKGROUND Photodynamic therapy (PDT) is alternative method for treating malignant tumors based on the principle of photodynamic damage to tumor cells through a photochemical reaction. Because of its localized effect, photodynamic therapy has become a very popular alternative treatment for cancer. PDT in combination with other drugs has been reported to have synergistic effects on various chemotherapeutic drugs. Thus for this synergistic effect of photodynamic therapy in combination with various chemotherapeutic drugs has gained the major interests to the scientists in recent days. Studies have been carried out to treat various ailments like cancer with this combination therapy. However, PDT in combination with biologically active natural product has not yet been studied in detail. One of the natural products which have been used as a folk medicine for many centuries is propolis. It is a resinous hive product collected from various plant materials by honeybees. It is reported to exhibit several biological activities. METHODS In this study, we focused on the effect of propolis and radachlorin-mediated PDT on human head and neck cancer cells AMC-HN-4. After the administration of propolis and radachlorin followed by laser irradiation, the viability of AMC-HN-4 cells was analyzed using MTT assay. The cells were also stained with Hoechst 33342 and propidium iodide (PI) for morphological observations. For more detailed evaluation and observation, flowcytometric analysis and western blotting were also carried out after congruent treatment process. RESULTS From the result it was found that the proliferation of AMC-HN-4 cells was inhibited by propolis. The inhibition of cell proliferation was increased when the cells were treated in combination. The rate of cell death was also increased in combination. The expressions of different proteins related to apoptosis were also regulated significantly. CONCLUSIONS Thus the results of this study indicate that the apoptosis and anti-proliferation efficacy of propolis were significantly enhanced in combination therapy, compared to the individual treatment of PDT or propolis.

Low-level laser therapy with 850 nm recovers salivary function via membrane redistribution of aquaporin 5 by reducing intracellular Ca 2+ overload and ER stress during hyperglycemia

The overall goal is to study the effect of low-level laser therapy (LLLT) on membrane distribution of major water channel protein aquaporin 5 (AQP5) in salivary gland during hyperglycemia. Par C10 cells treated with high glucose (50 mM) showed a reduced membrane distribution of AQP5. The functional expression of AQP5 was downregulated due to intracellular Ca2+ overload and ER stress. This reduction in AQP5 expression impairs water permeability and therefore results in hypo-salivation. A reduced salivary flow was also observed in streptozotocin (STZ)-induced diabetic mice model and the expression of AQP5 and phospho-AQP5 was downregulated. Low-level laser treatment with 850 nm (30 mW, 10 min = 18 J/cm2) reduced ER stress and recovered AQP5 membrane distribution via serine phosphorylation in the cells. In the STZ-induced diabetic mouse, LLLT with 850 nm (60 J/cm2) increased salivary flow and upregulated of AQP5 and p-AQP5. ER stress was also reduced via downregulation of caspase 12 and CHOP. In silico analysis confirmed that the serine 156 is one of the most favorable phosphorylation sites of AQP5 and may contribute to the stability of the protein. Therefore, this study suggests high glucose inhibits phosphorylation-dependent AQP5 membrane distribution. High glucose induces intracellular Ca2+ overload and ER stress that disrupt AQP5 functional expression. Low-level laser therapy with 850 nm improves salivary function by increasing AQP5 membrane distribution in hyperglycemia-induced hyposalivation.

Interstitial Laser Photocoagulation Using 980 nm Diode Laser in Benign Thyroid Nodule: A Feasibility Study

The aim of this study was to investigate the feasibility of using 980 nm diode laser for interstitial laser photocoagulation (ILP) before clinical application in benign thyroid nodule treatment. The bovine livers were cut into blocks to irradiate with 980 nm laser through the lumen of a 20-gauge spinal needle using a fiber optic guide. Laser irradiation was performed with the output power of 2 W and 3 W for 60, 120 and 180 seconds respectively. The liver blocks containing lesions were dissected along the axis of the fiber optic tracts and then cut transversely into slices. The thermal effect was evaluated by measuring the dimensions of the zone of coagulation necrosis. We present a case treated with 980 nm diode laser for the benign large thyroid nodule. All the irradiated areas zone measured in the gross specimens were 5.5 mm ± 1.4 mm (2 W, 60 s), 6.9 mm ± 1.4 mm (2 W, 120 s), 7.3 mm ± 0.5 mm (2 W, 180 s), 8.8 mm ± 2.2 mm (3 W, 60 s), 9.2 mm ± 0.8 mm (3 W, 120 s), 12.5 mm ± 4.1 mm (3 W, 180 s) respectively. The transverse diameter was as 5.1 mm ± 0.5 mm (2 W, 60 s), 6.1 mm ± 0.2 mm (2 W, 120 s), 9.9 mm ± 2.5 mm (2 W, 180 s), 6.2 mm ± 1.8 mm (3 W, 60 s), 7.7 mm ± 1.2 mm (3 W, 120 s), 8.8 mm ± 0.7 mm (3 W, 180 s) respectively. ILP was well tolerated and there was no complication. Interstitial laser photocoagulation with 980 nm diode laser induces well-defined tissue ablation correlated with energy parameters in bovine liver tissue and therefore, could be an efficient therapeutic tool in benign thyroid nodular disease.