Multi-Wavelength Photobiomodulation Ameliorates Sodium Iodate-Induced Age-Related Macular Degeneration in Rats.

Age-related macular degeneration (AMD) is a global health challenge. AMD causes visual impairment and blindness, particularly in older individuals. This multifaceted disease progresses through various stages, from asymptomatic dry to advanced wet AMD, driven by various factors including inflammation and oxidative stress. Current treatments are effective mainly for wet AMD; the therapeutic options for dry AMD are limited. Photobiomodulation (PBM) using low-energy light in the red-to-near-infrared range is a promising treatment for retinal diseases. This study investigated the effects of multi-wavelength PBM (680, 780, and 830 nm) on sodium iodate-induced oxidatively damaged retinal tissue. In an in vivo rat model of AMD induced by sodium iodate, multi-wavelength PBM effectively protected the retinal layers, reduced retinal apoptosis, and prevented rod bipolar cell depletion. Furthermore, PBM inhibited photoreceptor degeneration and reduced retinal pigment epithelium toxicity. These results suggest that multi-wavelength PBM may be a useful therapeutic strategy for AMD, mitigating oxidative stress, preserving retinal integrity, and preventing apoptosis.

Evaluation of the safety and efficacy of long pulsed Nd: YAG laser in the treatment of vascular lesions in vivo.

Long-pulsed neodymium:yttrium-aluminum-garnet (Nd:YAG) lasers have recently been used for the treatment of vascular lesions refractory to conventional vascular lasers. The aim of this study was to evaluate the clinical efficacy and safety of long-pulsed Nd:YAG laser treatment for vascular disorders. Laser irradiation was performed using two approaches: the 532 nm Nd:YAG laser was used to irradiate the dorsal skin fold in mice and the 1064 nm Nd:YAG laser was used to irradiate the leg of mice without skin incision. The specimens were observed immediately after laser treatment using a laser Doppler perfusion imaging system. Red blood cell (RBC) extravasation and hemorrhage were observed using the hematoxylin and eosin stain. The diameter of blood vessel under 30 µm was disrupted with a laser pulse at a fluence of 12 J/cm2 and a wavelength of 532 nm regardless of pulse duration. The veins and arteries of approximately 1 mm in size were ablated with laser pulses at a fluence of 140 J/cm2 and above and a wavelength of 1064 nm. Selective photopyrolysis can be achieved with either 532- or 1064 nm Nd:YAG laser pulses in vascular diseases based on the depth and size of the vessel.

Development of Intraoperative Near-Infrared Fluorescence Imaging System Using a Dual-CMOS Single Camera.

We developed a single-camera-based near-infrared (NIR) fluorescence imaging device using indocyanine green (ICG) NIR fluorescence contrast agents for image-induced surgery. In general, a fluorescent imaging system that simultaneously provides color and NIR images uses two cameras, which is disadvantageous because it increases the imaging head of the system. Recently, a single-camera-based NIR optical imaging device with quantum efficiency partially extended to the NIR region was developed to overcome this drawback. The system used RGB_NIR filters for camera sensors to provide color and NIR images simultaneously; however, the sensitivity and resolution of the infrared images are reduced by 1/4, and the exposure time and gain cannot be set individually when acquiring color and NIR images. Thus, to overcome these shortcomings, this study developed a compact fluorescent imaging system that uses a single camera with two complementary metal-oxide semiconductor (CMOS) image sensors. Sensitivity and signal-to-background ratio were measured according to the concentrations of ICG solution, exposure time, and camera gain to evaluate the performance of the imaging system. Consequently, the clinical applicability of the system was confirmed through the toxicity analysis of the light source and in vivo testing.

Photobiomodulation Attenuated Cognitive Dysfunction and Neuroinflammation in a Prenatal Valproic Acid-Induced Autism Spectrum Disorder Mouse Model.

Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by social communication and interaction disorders, as well as repetitive and restrictive behaviors. To date, no effective treatment strategies have been identified. However, photobiomodulation (PBM) is emerging as a promising treatment for neurological and neuropsychiatric disorders. We used mice exposed to valproic acid (VPA) as a model of ASD and found that pathological behavioral and histological changes that may have been induced by VPA were attenuated by PBM treatment. Pregnant mice that had been exposed to VPA were treated with PBM three times. Thereafter, we evaluated the offspring for developmental disorders, motor function, hyperactivity, repetitive behaviors, and cognitive impairment. PBM attenuated many of the pathological behaviors observed in the VPA-induced ASD mouse model. In addition, pathophysiological analyses confirmed that the increase in activated microglia and astrocytes observed in the VPA-induced ASD mouse model was attenuated by PBM treatment. This suggests that PBM can counteract the behavioral changes caused by neuroinflammation in ASD. Therefore, our data show that PBM has therapeutic potential and may reduce the prevalence of neurodevelopmental disorders such as ASD.

Combined treatment of low-level laser therapy and phloroglucinol for inhibition of fibrosis.

BACKGROUND AND OBJECTIVES: Fibrosis is a highly prevalent disease, which is responsible for 45% of deaths through pathological effects in developed countries. Previous studies have reported that low-level laser therapy (LLLT) can modulate fibrotic activity, but significant enhancement of therapeutic efficacy is still required for clinical translation. The aim of this study is to evaluate the feasible effect of LLLT combined with phloroglucinol (PHL) on the inhibition of fibrosis in vitro. STUDY DESIGN/MATERIALS AND METHODS: NIH/3T3 murine embryonic fibroblasts cells were cultured and transforming growth factor-ß1 (TGF-ß1) was treated for transition of fibroblasts. After TGF-ß1 treatment, LLLT and PHL were used, respectively, and in combination to suppress fibrosis. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and BrdU assays were performed to estimate the cell viability and proliferation. To evaluate the expression of fibrotic markers, we used confocal immunofluorescence and western blot. RESULTS: When compared with respectively treated groups, the group with the combined treatment of LLLT and PHL significantly reduced cell viability and proliferation. Immunofluorescence staining showed that the combined group minimized more α-smooth muscle actin (α-SMA) and type I collagen than the other groups. Western blot analysis showed that the combined treatment had significant decreases in α-SMA, TGF-ß1, and type I collagen. CONCLUSIONS: PHL-assisted LLLT may be an effective treatment to inhibit fibrosis due to its additive effects. The combined treatment has a potential to be an alternative treatment for fibrosis. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Effect of photobiomodulation therapy on neuronal injuries by ouabain: the regulation of Na, K-ATPase; Src; and mitogen-activated protein kinase signaling pathway.

BACKGROUND: To determine whether photobiomodulation (PBM) rescued the disruption of Na+/Ca2+ homeostasis and mitochondrial membrane potential by ouabain; the Na, K-ATPase inhibitor. For PBM in this study, a 660 nm LED array was used at energy densities of 0.78, 1.56, 3.12, 6.24, and 9.36 J/cm2. RESULTS: HCN-2 neuronal cells treated with ouabain showed loss of cell polarity, disrupted cell morphology, and decreased cell viability, which were improved after PBM treatment. We found that ouabain-induced Na, K-ATPase inhibition promoted activation of downstream signaling through Src, Ras, and mitogen-activated protein kinase (MAPK), which were suppressed after PBM treatment. This provided evidence of Na, K-ATPase α-subunit inactivation and intracellular Ca2+ increase. In response to ouabain, we observed activation of Src and MAPK by Na, K-ATPase, decreased mitochondrial membrane potential, and Na+-dependent Ca2+ increases, which were restored by PBM treatment. CONCLUSIONS: This study demonstrated that Na+/K+ imbalance could be regulated by PBM treatment in neuronal cells, and we suggest that PBM is a potential therapeutic tool for Na, K-ATPase targeted neuronal diseases.

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.

Enhanced survival of ischemic skin flap by combined treatment with bone marrow-derived stem cells and low-level light irradiation.

The aim of this study is to examine the enhanced survival effect of ischemic skin flap by combined treatment with bone marrow-derived stem cells (BMSCs) and low-level light irradiation (LLLI). The neovasculogenic effect of BMSCs induced by LLLI was detected using a wound healing and tube formation assay. ICR mice were divided into four groups: control group, LLLI group, BMSCs group, and combine-treated group. The percentage of skin flap necrosis area was calculated on the seventh post-operative day. Specimens were harvested for histologic analyses. LLLI promoted BMSC migration and tube formation. The flap survival rate of combined treated group was significantly higher than that of the control group. Histologic results demonstrated a significant increase in neovascularization in the combined treatment group. This study demonstrates that combination treatment of BMSCs and LLLI could enhance the survival of ischemic skin flap in a mouse model.

Adipose-derived stem cell spheroid treated with low-level light irradiation accelerates spontaneous angiogenesis in mouse model of hindlimb ischemia.

BACKGROUND AIMS: We investigated whether low-level light irradiation (LLLI) before adipose-derived stromal cells (ASCs) spheroid transplantation improved hind-limb functional recovery by stimulation of angiogenesis. METHODS: The spheroid, composed of ASCs, was irradiated with low-level light and expressed angiogenic factors, including vascular endothelial growth factor and basic fibroblast growth factor. From immunochemical staining analysis, the spheroid of ASCs included CD31+, KDR+ and CD34+, whereas monolayer-cultured ASCs were negative for these markers. To evaluate the therapeutic effect of the ASC spheroid treated with LLLI in vivo, phosphate-buffered saline, monolayer ASCs, LLLI-monolayer ASCs, spheroid ASCs and LLLI-spheroid ASCs were transplanted into a hind-limb ischemia model. RESULTS: The LLLI-spheroid ASCs transplanted into the hind-limb ischemia differentiated into endothelial cells and remained differentiated. Transplantation of LLLI-spheroid ASCs into the hind-limb ischemia significantly elevated the density of vascular formations through angiogenic factors released by the ASCs and enhanced tissue regeneration at the lesion site. Consistent with these results, the transplantation of LLLI-spheroid ASCs significantly improved functional recovery compared with ASC or spheroid ASC transplantation and PBS treatment. CONCLUSIONS: These findings suggest that transplantation of ASC spheroid treated with LLLI may be an effective stem cell therapy for the treatment of hind-limb ischemia and peripheral vascular disease.

Human adipose-derived stem cell spheroid treated with photobiomodulation irradiation accelerates tissue regeneration in mouse model of skin flap ischemia.

Skin flap grafting is a form of transplantation widely used in plastic surgery. However, ischemia/reperfusion injury is the main factor which reduces the survival rate of flaps following grafting. We investigated whether photobiomodulation (PBM) precondition prior to human adipose-derived stromal cell (hASC) spheroid (PBM-spheroid) transplantation improved skin tissue functional recovery by the stimulation of angiogenesis and tissue regeneration in skin flap of mice. The LED had an emission wavelength peaked at 660 ± 20 nm (6 J/cm2, 10 mW/cm2). The expression of angiogenic growth factors in PBM-spheroid hASCs was much greater than that of not-PBM-treated spheroid or monolayer-cultured hASCs. From immunochemical staining analysis, the hASCs of PBM-spheroid were CD31+, KDR+, and CD34+, whereas monolayer-cultured hASCs were negative for these markers. To evaluate the therapeutic effect of hASC PBM-spheroid in vivo, PBS, monolayer-cultured hASCs, and not-PBM-spheroid were transplanted into a skin flap model. The animals were observed for 14 days. The PBM-spheroid hASCs transplanted into the skin flap ischemia differentiated into endothelial cells and remained differentiated. Transplantation of PBM-spheroid hASCs into the skin flap ischemia significantly elevated the density of vascular formations through angiogenic factors released by the skin flap ischemia and enhanced tissue regeneration at the lesion site. Consistent with these results, the transplantation of PBM-spheroid hASCs significantly improved functional recovery compared with PBS, monolayer-cultured hASCs, and not-PBM-spheroid treatment. These findings suggest that transplantation of PBM-spheroid hASCs may be an effective stem cell therapy for the treatment of skin flap ischemia.

Angiogenic Synergistic Effect of Adipose-Derived Stromal Cell Spheroids with Low-Level Light Therapy in a Model of Acute Skin Flap Ischemia.

Human adipose-derived mesenchymal stem cells (hASCs) are an attractive cell source for tissue engineering. However, one obstacle to this approach is that the transplanted hASC population can decline rapidly in the recipient tissue. The aim of this study was to investigate the effects of low-level light therapy (LLLT) on transplanted spheroid hASCs in skin flaps of mice. hASCs were cultured in monolayers or spheroids. LLLT, hASCs, spheroids and spheroids transplanted with LLLT were applied to the skin flaps. Healing of the skin flaps was assessed by gross evaluation and by hematoxylin and eosin staining and elastin van Gieson staining. Compared with the spheroid group, skin flap healing was enhanced in the spheroid + LLLT group, including the neovascularization and regeneration of skin appendages. The survival of hASCs was enhanced by decreased apoptosis of hASCs in the skin flaps of the spheroid + LLLT group. The secretion of growth factors was stimulated in the spheroid + LLLT group compared with the ASC and spheroid groups. These data suggest that LLLT was an effective biostimulator of spheroid hASCs in the skin flaps, enhancing the survival of hASCs and stimulating the secretion of growth factors.

Safety assessment of trans-tympanic photobiomodulation.

We evaluated functional and morphological changes after trans-tympanic laser application using several different powers of photobiomodulation (PBM). The left (L) ears of 17 rats were irradiated for 30 min daily over 14 days using a power density of 909.1 (group A, 5040 J), 1136.4 (group B, 6300 J), and 1363.6 (group C, 7560 J) mW/cm(2). The right (N) ears served as controls. The safety of PBM was determined by endoscopic findings, auditory brainstem response (ABR) thresholds, and histological images of hair cells using confocal microscopy, and light microscopic images of the external auditory canal (EAC) and tympanic membrane (TM). Endoscopic findings revealed severe inflammation in the TM of C group; no other group showed damage in the TM. No significant difference in ABR threshold was found in the PBM-treated groups (excluding the group with TM damage). Confocal microscopy showed no histological difference between the AL and AN, or BL and BN groups. However, light microscopy showed more prominent edema, inflammation, and vascular congestion in the TM of BL ears. This study found a dose-response relationship between laser power parameters and TM changes. These results will be useful for defining future allowance criteria for trans-tympanic laser therapies.

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.

Adipose-derived stromal cell cluster with light therapy enhance angiogenesis and skin wound healing in mice.

Human adipose-derived mesenchymal stem cells (hASCs) are attractive cell source for skin tissue engineering. The aim of this study was to investigate the effects of low-level light therapy (LLLT) on transplanted cluster hASC in a skin wound animal model. The hASCs were cultured in monolayer or clusters. The LLLT, hASCs, hASC clusters, and hASC clusters transplantation with LLLT (cluster + LLLT) were applied to the wound bed in athymic mice. Wound healing was assessed by gross evaluation and by hematoxylin and eosin staining, and elastin van gieson histochemistry. The survival, differentiation, and secretion of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), and hepatocyte growth factor (HGF) of the cluster ASC were evaluated by immunohistochemistry and Western blotting. The cluster + LLLT group enhanced the wound healing, including neovascularization and regeneration of skin appendages, compared with the cluster group. The secretion of growth factors was stimulated in the cluster + LLLT group compared with the ASCs and cluster group. These data suggest that LLLT is an effective biostimulator of cluster hASCs in wound healing that enhances the survival of hASCs and stimulates the secretion of growth factors in the wound bed.

Circumferential irradiation for interstitial coagulation of urethral stricture.

An optical diffuser was developed to achieve radially uniform light irradiation by micro-machining helical patterns on the fiber surface for endoscopically treating urethral stricture. Spatial emission from the diffuser was evaluated by goniometric measurements. A computational model was developed to predict spatio-temporal heat distribution during the interstitial coagulation. The fabricated diffuser yielded circumferential light distribution with slightly concentrated energy at the proximal end. Both simulation and tissue testing demonstrated approximately 1-mm coagulation thickness at 6 W for 10 sec with 1470 nm. The proposed optical diffuser may be a feasible tool to treat the urethral stricture in a uniform manner.

Prevention of skin flap necrosis by use of adipose-derived stromal cells with light-emitting diode phototherapy.

BACKGROUND AIMS: The aim of this study was to investigate the effects of low-level light therapy (LLLT) on transplanted human adipose-derived mesenchymal stromal cells (ASCs) in the skin flap of mice. METHODS: LLLT, ASC transplantation and ASC transplantation with LLLT (ASC + LLLT) were applied to the skin flap. Immunostaining and Western blot analysis were performed to evaluate cell survival and differentiation and secretion of vascular endothelial growth factor and basic fibroblast growth factor by the ASCs. Vascular regeneration was assessed by means of immunostaining in addition to hematoxylin and eosin staining. In the ASC + LLLT group, the survival of ASCs was increased as the result of the decreased apoptosis of ASCs. RESULTS: The secretion of growth factors was higher in this group as compared with ASCs alone. ASCs contributed to tissue regeneration through vascular cell differentiation and secretion of angiogenic growth factors. The ASC + LLLT group displayed improved treatment efficacy including neovascularization and tissue regeneration compared with ASCs alone. Transplanting ASCs to ischemic skin flaps improved therapeutic efficacy for ischemia treatment as the result of enhanced cell survival and paracrine effects. CONCLUSIONS: These data suggest that LLLT is an effective biostimulator of ASCs in vascular regeneration, which enhances the survival of ASCs and stimulates the secretion of growth factors in skin flaps.

Vascular regeneration effect of adipose-derived stem cells with light-emitting diode phototherapy in ischemic tissue.

The objective of this study was to investigate the effects on the vascular regeneration of adipose-derived stem cells (ASCs) by using red light-emitting diode (LED) irradiation in ischemic hind limbs. Low-level light therapy (LLLT) has been shown to enhance proliferation and cytokine secretion of a number of cells. ASCs are an attractive cell source for vascular tissue engineering. This approach is hindered because transplanted ASCs decline rapidly in the recipient tissue. Ischemic hind limbs were treated with LLLT from an LED array (660 nm) at an irradiance of 50 mW/cm(2) and a radiant exposure of 30 J/cm(2). LLLT, ASC transplantation, and ASC transplantation with LLLT (ASC + LLLT) were applied to ischemic limbs, and cell survival and differentiation, and secretion of vascular endothelial growth factor and basic fibroblast growth factor of the ASCs were evaluated by immunostaining and Western blot analyses. Vascular regeneration was assessed by immunostaining and hematoxylin and eosin staining. In the ASC + LLLT group, the survival of ASCs was increased due to the decreased apoptosis of ASCs. The secretion of growth factors was stimulated in this group compared with ASCs alone. The ASC + LLLT group displayed improved treatment efficacy including neovascularization and tissue regeneration compared with ASCs alone. In particular, quantitative analysis of laser Doppler blood perfusion image ratio showed that blood perfusion was enhanced significantly (p < 0.05) by ASC + LLLT treatment. These data suggest that LLLT is an effective biostimulator of ASCs in vascular regeneration, which enhances the survival of ASCs and stimulates the secretion of growth factors in ischemic limbs.

Enhancement of cytotoxic effect on human head and neck cancer cells by combination of photodynamic therapy and sulforaphane.

Photodynamic therapy (PDT) is a method to treat cancers using photosensitizer and light. PDT has been tried for several tumors. However, the clinical applications are limited by the toxicity of photosensitizer and narrow effect. Sulforaphane (SFN) is a material of isothiocyanate group and known to have anticancer effect. We evaluated the cytotoxic effect of PDT combined with SFN on human head and neck cancer cells. We measured the cell viability, extent of apoptosis and necrosis, reactive oxygen species (ROS) generation and caspase activation. Cell viability was decreased significantly by combination treatment. Cellular apoptosis and necrosis were increased in combination treatment compared to SFN or PDT. ROS generation was also higher in combination treatment than single treatment. In combination treatment group, apoptosis and necrosis were decreased by administration of sodium azide (SA) which is scavenger of ROS. Increased caspase activation in combination treatment was also inhibited by SA. Combination of PDT and SFN led to enhanced cytotoxic effect on head and neck cancer cells. Combination treatment promoted the ROS generation, which induced cell death through activation of caspase pathway.

CXCR4 and PTEN are involved in the anti-metastatic regulation of anethole in DU145 prostate cancer cells.

Anethole has been known to have chemopreventive activities as a suppressor of the incidence and multiplicity of both invasive and noninvasive carcinomas. The goal of this study was to understand the anti-metastatic effect of anethole through C-X-C chemokine receptor type 4 (CXCR4)/tumor suppressor phosphatase and tensin homologue (PTEN) axis in DU145 prostate cancer cells. Anethole reduced both of the RNA level and the protein level of CXCR4 in a dose-dependent manner without cytotoxicity. Anethole also reduced the expression of CXCR4 and prolonged the expression of PTEN in DU145 prostate cancers. The phosphorylation of AKT and phosphatidylinositol-3kinase (PI3K) were decreased with anethole. The inhibition metastatic effect of anethole was arisen from down-regulating CXCR4 and up-regulating PTEN. Morphologically, anethole significantly inhibited the invasion of DU145 cell and down-regulated the activities of matrix-metalloproteinase (MMPs) in a dose-dependent manner. However, anethole didnot decrease the phosphorylation of PI3K and AKT while PTEN was silenced. Furthermore, the CXCR4 inhibition of anethole was not caused to proteasomal or lysosomal of CXCR4. Taken together, anethole demonstrated to act as the CXCR4 antagonist and as the PTEN activator which resulted to PI3K/AKT-mediated inhibition of the metastatic prostate cancer progressions.

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.