Low-level laser therapy with 850 nm recovers salivary function via membrane redistribution of aquaporin 5 by reducing intracellular Ca2+ 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.

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.

Cellular uptake of 9-hydroxypheophorbide-α and its photoactivation to induce ER stress-related apoptosis in human cervical cancer cells.

The 9-hydroxypheophorbide-α (9-HPbD) is a chlorophyll derivative and was found to be very effective for photodynamic therapy of tumor cells. The current study investigates uptake, retention, and intracellular localization of 9-HPbD by HeLa, human cervical cancer cells via fluorescence spectrophotometry and confocal laser scanning microscopy, and its photodynamic effect against human cervical carcinoma cell. HeLa cells exposed to 9-HPbD exhibited a linear uptake of photosensitizer during the first 12 h, and after removal of 9-HPbD, cell fluorescence was observed to decrease gradually over the next 12 h. Cells treated with 9-HPbD and stained with a panel of organelle-specific fluorescence probes (MitoTracker, LysoTracker, and ER-Tracker) revealed an intracellular fluorescence distribution restricted to cytoplasmic compartments with no detectable fluorescence in the nucleus. The 9-HPbD showed cytotoxicity effect against HeLa cells in 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Endoplasmic reticulum (ER) disruption and cellular calcium dynamics also showed a photoactivation followed by cell death. The apoptotic effect of 9-HPbD was confirmed by caspase 3 activity study and immunofluorescence study of caspase 12. Morphological observation through the transmission electron microscopy and scanning electron microscopy also confirmed that 9-HPbD can induce apoptosis in HeLa cells. Therefore, it can be concluded that maximum uptake and clearance time of 9-HPbD was 12 h with endoplasmic reticulum as the major organelle site in cellular uptake, and 9-HPbD can induce apoptosis in HeLa cells through ER stress-related pathways via activation of caspase 12.

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.

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.

Condurango-glycoside-A fraction of Gonolobus condurango induces DNA damage associated senescence and apoptosis via ROS-dependent p53 signalling pathway in HeLa cells.

Gonolobus condurango plant extract is used as an anticancer drug in some traditional systems of medicine including homeopathy, but it apparently lacks any scientific validation. Further, no detailed study is available to suggest whether condurango-glycoside-A (CGA), a major ingredient of condurango serves as a potent anticancer compound. Therefore, we investigated apoptosis-inducing ability of CGA against cervix carcinoma cells (HeLa). ß-galactosidase-activity and DNA damage were critically studied at different time points; while induced DNA-damage was observed at 9-12th hours, senescence of cells appeared at a later stage (18th hour after CGA treatment), implicating thereby a possible role of DNA damage in inducing pre-mature cell senescence. Concurrently, the number of cells undergoing apoptosis increased along with increase in reactive oxygen species (ROS) generation. Expression of p53 was also up-regulated, indicating that apoptosis could have been mediated through p53 pathway. DCHFDA (4',6-Diamidino-2-phenylindole dihydrochloride) assay, acridine orange/ethidium bromide staining and annexin V/PI assay results collectively confirmed that apoptosis was induced by increased ROS generation. Reduction in proliferation of cells was further evidenced by the cell cycle arrest at G0/G1 stage. Expression profiles of certain relevant genes and proteins like p53, Akt, Bcl-2, Bax, cytochrome c and caspase 3 also provided evidence of ROS mediated p53 up-regulation and further boost in Bax expression and followed by cytochrome c release and activation of caspase 3. Overall results suggest that CGA initiates ROS generation, promoting up-regulation of p53 expression, thus resulting in apoptosis and pre-mature senescence associated with DNA damage.

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

BACKGROUND AND OBJECTIVE: 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. MATERIALS AND METHODS: Combination of PDT and genistein has been studied against human thyroid cancer cells SNU 80. Cells were treated with genistein and different concentration of photofrin in PDT singly and conjointly. Viability of SNU 80 cells was analyzed using MTT assay. The cells were stained with Hoechst 33342 and propidium iodide (PI) for morphological observations. Changes in mitochondrial membrane potential and generation of reactive oxygen species (ROS) were also studied by confocal microscopy. Western blot analysis were also performed to find out the expressions of different pro- and anti-apoptotic proteins. RESULTS: From the result, the combination of genistein and photofrin mediated PDT enhanced the apoptotic effect against SNU 80 cells. Proliferation of the cells was inhibited and the mitochondrial membrane depolarisation was observed. ROS level were also increased in combination treatment. The expressions of Caspase 3, Caspase 9, cytochrome c, Caspase 8, Caspae 12 and other apoptosis related proteins were also modified. CONCLUSION: Thus PDT can induce apoptosis in thyroid cancer cells singly or in combination with genistein. But in combination treatment, the efficacy of inducing apoptosis in SNU 80 cells is much higher than that of individual treatment with genistein or PDT.

Ameliorative potentials of Syzygium jambolanum extract against arsenic-induced stress in L6 cells in vitro.

OBJECTIVE: To determine the ameliorative potentials of Syzygium jambolanum (SJ) extract in L6 skeletal muscle cells in regard to arsenic-induced impairment of optimum glucose homeostasis and improper functioning of mitochondria. METHODS: Several study parameters like glucose level and mitochondrial functioning through indexes of pyruvate kinase, glucokinase and mitochondrial membrane potential were assessed. The expression of the relevant marker proteins and mRNAs like glucose transporter 4 (GLUT4), insulin receptor substrate 1 (IRS1), IRS2 and glucokinase for tracking down the signalling cascade were critically analyzed. RESULTS: Introduction of SJ extract could bring about positive modulation of various markers, by acting on GLUT4, thereby bringing about an attenuation of the arsenite-induced toxic conditions in L6 cells. CONCLUSION: Syzygium jambolanum extract has considerable ameliorating potentials against arsenic-induced glucose imbalance and stress and has possibility of therapeutic use in the management of arsenic-induced toxicity including hyperglycemia.

Thujone-Rich Fraction of Thuja occidentalis Demonstrates Major Anti-Cancer Potentials: Evidences from In Vitro Studies on A375 Cells.

CRUDE ETHANOLIC EXTRACT OF THUJA OCCIDENTALIS (FAM: Cupressaceae) is used as homeopathic mother tincture (TOΦ) to treat various ailments, particularly moles and tumors, and also used in various other systems of traditional medicine. Anti-proliferative and apoptosis-inducing properties of TOΦ and the thujone-rich fraction (TRF) separated from it have been evaluated for their possible anti-cancer potentials in the malignant melanoma cell line A375. On initial trial by S-diphenyltetrazolium bromide assay, both TOΦ and TRF showed maximum cytotoxic effect on A375 cell line while the other three principal fractions separated by chromatography had negligible or no such effect, because of which only TRF was further characterized and subjected to certain other assays for determining its precise anti-proliferative and apoptotic potentials. TRF was reported to have a molecular formula of C(10)H(16)O with a molecular weight of 152. Exposure of TRF of Thuja occidentalis to A375 cells in vitro showed more cytotoxic, anti-proliferative and apoptotic effects as compared with TOΦ, but had minimal growth inhibitory responses when exposed to normal cells (peripheral blood mononuclear cell). Furthermore, both TOΦ and TRF also caused a significant decrease in cell viability, induced inter-nucleosomal DNA fragmentation, mitochondrial transmembrane potential collapse, increase in ROS generation, and release of cytochrome c and caspase-3 activation, all of which are closely related to the induction of apoptosis in A375 cells. Thus, TRF showed and matched all the anti-cancer responses of TOΦ and could be the main bio-active fraction. The use of TOΦ in traditional medicines against tumors has, therefore, a scientific basis.

Potentized homeopathic drug Arsenicum Album 30C positively modulates protein biomarkers and gene expressions in Saccharomyces cerevisae exposed to arsenate.

OBJECTIVE: This study examines if homeopathic drug Arsenicum Album 30C (Ars Alb 30C) can elicit ameliorative responses in yeast (Saccharomyces cerevisiae) exposed to arsenate. METHODS: The yeast S. cerevisiae 699 was cultured in a standard yeast extract peptone dextrose broth medium. It was exposed to the final concentration of 0.15 mmol/L arsenate for two intervals, 1 h and 2 h, respectively. The cell viability was determined along with the assessment of several toxicity biomarkers such as catalase (CAT), superoxide dismutase (SOD), total thiol (GSH) and glucose-6-phosphate dehydrogenase (G6PDH), lipid peroxidation, protein carbonylation and DNA damage. Reactive oxygen species (ROS) accumulation, expressions of relevant stress transcription activators like Yap-1 and Msn 2, and mRNA expression of yeast caspase-1 (Yca-1) were also measured. RESULTS: Treatment of arsenate increased lipid peroxidation, protein carbonylation, DNA damage, ROS accumulation and expressions of Yap-1, Msn 2 and Yca-1 and decreased GSH, G6PDH, CAT and SOD. Ars Alb 30C administration decreased lipid peroxidation, protein carbonylation, DNA damage, ROS formation and Msn 2 and Yca-1 expressions and increased cell viability, GSH, G6PDH, CAT and SOD significantly (P<0.05), except for a slight increase in Yap-1 expression. CONCLUSION: Ars Alb 30C triggers ameliorative responses in S. cerevisiae exposed to arsenate.

Tolerance of arsenate-induced stress in Aspergillus niger, a possible candidate for bioremediation.

The arsenate tolerance limit in wild-type Aspergillus niger was determined. Because of its high tolerance, toxic effects of arsenate concentrations ranging from 25 to 100mg/L were investigated in regard to growth, intracellular thiols, proline and malondialdehyde (MDA) contents of wild-type A. niger. Cellular arsenate uptake was analyzed. Activities of catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR) and succinate dehydrogenase (SDH) were assayed. Growth of A. niger increased at 25mg/L arsenate, and it survived up to 100mg/L. MDA, intracellular thiol and proline contents increased up to a certain level. Activities of GR, SOD and CAT declined following a rise at low concentration(s); SDH activity decreased gradually with increased arsenate stress. Results indicated that A. niger had high arsenate uptake potential and could tolerate oxidative stress by manipulating its anti-oxidative defense mechanism, a property that may be exploited for removal of arsenate from contaminated aqua-environment.

Light-emitting diode irradiation using 660 nm promotes human fibroblast HSP90 expression and changes cellular activity and morphology.

We evaluated changes in cell viability and morphology in response to low-level light irradiation and underlying variations in the levels of heat shock proteins (HSPs). Human fibroblasts were irradiated with a light-emitting diode (LED) array at 660 nm (50 mW for 15, 30, and 60 minutes). Cell viability and morphological changes were evaluated via epifluorescence analysis; we also assessed cell viability and length changes. The expression levels of adenosine triphosphate (ATP) and various HSPs (HSP27, 60, 70, and 90) were analyzed by immunohistochemical staining, Western blotting and microarray analysis. After LED irradiation, cellular viability and morphology changed. Of the several HSPs analyzed, the HSP90 level increased significantly, suggesting that this protein played roles in the morphological and cellular changes. Thus, low-level irradiation triggered cellular changes mediated by increased HSP90 expression; this may explain why skin irradiation enhances wound-healing.

In vitro studies demonstrate anticancer activity of an alkaloid of the plant Gelsemium sempervirens.

The chemical structure of the main fluorescenting compound in the ethanolic extract (mother tincture) of the American yellow jasmine, Gelsemium sempervirens, was determined by employing (1)H nuclear magnetic resonance (NMR), (13)C NMR, mass spectroscopy, high-performance liquid chromatography (HPLC), correlation spectroscopy (COSY), and Fourier transform infrared (FTIR) spectroscopy analyses. Spectrofluorometric analysis has been made of the mother tincture and its agitated serial dilutions (up to 12th potency) prepared according to a homeopathic procedure in which serial, agitated dilutions were made separately in glass and polypropylene containers. The succussions were made by employing three different modes: hand jerk, sonication, and vortexing. The chemical formula of scopoletin, the main fluorescent compound, was determined to be C(10)H(8)O(4) having a molecular weight of 192.17. Significant differences were noted between the remedies prepared in the two types of containers. Further, a comparison between any two methods of agitation revealed significant differences in fluorometric data of remedies at certain potency levels. The biological (anticancer) action of the crude extract, the alkaloid scopoletin, and 2C potency of Gelsemium sp were tested in vitro on the HeLa cell line through fluorescence microscopy, the 3(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, and fluorescent activated cell sorting (FACS). The role of nanoparticles presumably derived from the containers, their orientation, and their interaction with the starting substance during the dynamization process initiated by different modes of agitation could possibly be attributed to the differences noted in the fluorometric data of potencies prepared in the two types of containers and among the three different means of succussion tested.

Sulforaphene Synergistically Sensitizes Cisplatin via Enhanced Mitochondrial Dysfunction and PI3K/PTEN Modulation in Ovarian Cancer Cells.

AIM: To explore if a natural isothiocyanate, sulforaphene (SFE), sensitizes ovarian cancer cells to the chemotherapy drug cisplatin (CDDP). MATERIALS AND METHODS: We studied reactive oxygen species (ROS), mitochondrial membrane depolarization and cell-cycle distribution in two ovarian cancer cell lines SKOV3 and SNU 8 treated with SFE and cisplatin. We further analyzed the expression of caspases 3, 8, and 9, Phosphoinositide 3-kinase (PI3K) and Phosphatase and tensin homolog (PTEN) by western blotting. RESULTS: SFE sensitized cells to cisplatin by enhancing ROS and mitochondrial membrane depolarization that released cytochrome c and activated caspase 9 and caspase 3 in the mitochondrial pathway. It also inhibited extrinsic pathway protein caspase 8, growth-related protein PI3K and further activated PTEN in combination with cisplatin. CONCLUSION: SFE synergistically inhibited proliferation and induced apoptosis of SKOV3 and SNU8 cells in combination with cisplatin by activating multiple apoptotic pathways. Therefore, we suggest sulforaphene as a chemo-enhancing adjuvant to improve the efficacy of cisplatin in ovarian cancer treatment.

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.

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.

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.

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.

[6]-Gingerol induces caspase 3 dependent apoptosis and autophagy in cancer cells: drug-DNA interaction and expression of certain signal genes in HeLa cells.

[6]-Gingerol, a pharmacologically important bioactive component of ginger, has been reported to have anti-hyperglycemic, anti-cancer and anti-oxidative properties, but mechanisms through which these are achieved are largely unclear. The present study focuses on apoptosis and autophagy, two key events of anti-cancer activity, in HeLa cells treated with [6]-gingerol. The treated cells showed several morphological changes, including externalization of phosphatidyl serine, degradation of DNA and increase in TUNEL positivity. Furthermore, there was depolarization of mitochondrial membrane potential, providing evidence of mitochondria mediated apoptosis. The expression of caspase 3 and PARP was increased in cells exposed to [6]-gingerol. Circular dichroism study for testing drug-DNA interaction with both calf thymus and nuclear DNA as target revealed that the drug had potential to bind with the nuclear DNA and induce conformational changes of DNA. The over-expression of NFkß, AKT and Bcl2 genes in cancer cells was down-regulated by [6]-gingerol treatment. On the other hand the expression levels of TNFα, Bax and cytochrome c were enhanced in [6]-gingerol treated cells. Thus, overall results suggest that [6]-gingerol has potential to bind with DNA and induce cell death by autophagy and caspase 3 mediated apoptosis.

Lycopodine from Lycopodium clavatum extract inhibits proliferation of HeLa cells through induction of apoptosis via caspase-3 activation.

Crude ethanolic extract of the plant Lycopodium clavatum has long been used in complementary and alternative medicine for treating various liver ailments and Alzheimer's disease. It has also been claimed to have potential anti-cancer properties in vivo in mice chronically fed liver carcinogens, p-dimethylamino azobenzene (initiator) and phenobarbital (promoter). Incidentally, crude ethanolic extract of Lycopodium clavatum is a mixture of some 201 alkaloids. In order to ascertain if any major fraction can be attributed to have pronounced anti-cancer effect, we examined this major fraction by eluting the crude extract in petroleum ether:ethyl aetate (17:3 vol/vol;) solvent and tried to understand its underlying mechanism. Studies on morphological changes, cell viability and cytotoxicity by microscopy and FACS, Western blot and immunofluorescence of Bcl-2, Bax, cytochrome c, caspase-3 were conducted. Lycopodine was found to induce chromatin condensation, inter-nucleosomal DNA fragmentation and enhanced cell population in sub-G1 region along with increase in reactive oxygen species generation and mitochondrial membrane potential depolarization, release of cytochrome c and activation of caspase-3 which are the events closely involved in apoptosis. An overall analysis of results showed that Lycopodine considerably inhibited growth of HeLa cells which indicates its potential use in chemotherapy.