The effects of Ce6-mediated sono-photodynamic therapy on cell migration,apoptosis and autophagy in mouse mammary 4T1 cell line

Purpose

Sono-Photodynamic therapy (SPDT) is an alternative therapy which claims to enhance the anti-cancer effects by combining sonodynamic therapy (SDT) with photodynamic therapy (PDT). In the present study, we investigated the effects of chlorin e6 (Ce6) mediated SPDT on migration, apoptosis and autophagy in mouse mammary 4T1 cancer cells, and its underlying mechanisms.

Materials

Cell migration was determined by wound healing assay. Apoptosis was analyzed using annexin V-PE/7-ADD staining as well as Hoechst 33342 staining. Changes of mitochondria membrane potential (MMP) was evaluated by flow cytometry. Formation of acidic vesicular organelles (AVOs) during autophagy was observed with fluorescence microscope by MDC staining. Immunofluorescence assays were performed to detect the co-localization of LC3 and Lamp2. Western blotting was employed to analyze the activity of the apoptosis related proteins Caspase-3, PARP, Bax and Bcl-2, as well as the autophagy associated processing of LC3-I to LC3-II and Beclin-1 expression.

Results

Ce6 mediated SPDT further enhanced cell migration inhibition, significantly triggered cell apoptosis, nuclear condensation and MMP drop. Cleaved Caspase-3 and PARP increased dramatically after Ce6-SPDT, accompanied by decreased Bcl-2 expression, while the expression of Bax remained stable. Additionally, AVOs formation, co-localization of LC3 and Lamp2 occurred following Ce6-SPDT and simultaneously accompanied by LC3-II processing and increased Beclin-1 expression.

Conclusions

Ce6-SPDT could enhance cell migration inhibition, and induce mitochondria-dependent apoptosis as well as autophagy in 4T1 cells.     

Sinoporphyrin sodium triggered sono-photodynamic effects on breast cancer both in vitro and in vivo

Sono-photodynamic therapy (SPDT) is a promising anti-cancer strategy. Briefly, SPDT combines ultrasound and light to activate sensitizers that produce mechanical, sonochemical and photochemical activities. Sinoporphyrin sodium (DVDMS) is a newly identified sensitizer that shows great potential in both sonodynamic therapy (SDT) and photodynamic therapy (PDT). In this study, we primarily evaluated the combined effects of SDT and PDT by using DVDMS on breast cancer both in vitro and in vivo. In vitro, DVDMS-SPDT elicits much serious cytotoxicity compared with either SDT or PDT alone by MTT and colony formation assays. 2′,7′-Dichlorodihydrofluo-rescein-diacetate (DCFH-DA) and dihydroethidium (DHE) staining revealed that intracellular reactive oxygen species (ROS) were significantly increased in groups given combined therapy. Terephthalic acid (TA) method and FD500-uptake assay reflected that cavitational effects and cell membrane permeability changes after ultrasound irradiation were also involved in the enhancement of combination therapy. In vivo, DVDMS-SPDT markedly inhibits the tumor volume and tumor weight growth. Hematoxylin-eosin staining and immunohistochemistry analysis show DVDMS-SPDT greatly suppressed tumor proliferation. Further, DVDMS-SPDT significantly inhibits tumor lung metastasis in the highly metastatic 4T1 mouse xenograft model, which is consistent well with the in vitro findings evaluated by transwell assay. Moreover, DVDMS-SPDT did not produces obvious effect on body weight and major organs in 4T1 xenograft model. The results suggest that by combination SDT and PDT, the sensitizer DVDMS would produce much better therapeutic effects, and DVDMS-SPDT may be a potential strategy against highly metastatic breast cancer.     

Nanosonosensitization by Using Copper–Cysteamine Nanoparticles Augmented Sonodynamic Cancer Treatment

Sonodynamic therapy (SDT), as a newly emerging and promising modality for cancer treatment, has been extensively investigated but with limited therapeutic outcome because of the absence of highly efficient sonosensitizer. Copper–cysteamine (Cu–Cy), as a new sensitizer, has been reported for oxidative therapy which can be activated with light, X‐ray, or microwave. Herein, for the first time, Cu–Cy nanoparticles are reported as new sonosensitizers for SDT on breast cancer treatment. Upon exposure of Cu–Cy nanoparticles to ultrasound, a large quantity of reactive oxygen species (ROS) are generated for cancer cell destruction with a high SDT efficiency to induce cell apoptosis and necrosis as observed in vitro. In vivo animal studies show a significant inhibition of tumor growth for the xenografts of 4T1 cancer cells with the combination of 0.75 mg kg⁻¹ Cu–Cy and ultrasound. Overall, the preliminary results show that Cu–Cy nanoparticles can significantly augment the levels of ROS induced by ultrasound, demonstrating Cu–Cy is a new kind of efficient sonosensitzers for SDT applications. Such therapeutic platform by integrating a noninvasive, highly safe, deep‐penetration ultrasound modality. and quickly developed versatile nanosensitizers for tumor eradication will facilitate SDT future clinical translation.     

Protoporphyrin IX-mediated sonodynamic action induces apoptosis of K562 cells

Objectives

The present study aims to investigate apoptosis of human leukemia K562 cells induced by protoporphyrin IX (PpIX)-mediated sonodynamic therapy (PpIX-SDT).

Methods

The uptakes of intracellular PpIX in K562 cells were detected by flow cytometry. The sub-cellular localization of PpIX was imaged by confocal microscope. The cytotoxic effect of PpIX-SDT was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenylter-trazolium bromide tetrazolium) assay. Apoptosis was evaluated by chromatin condensation with DAPI (4′-6-diamidino-2-phenylindole) staining, decrease of mitochondria membrane potential (MMP), re-distribution of Bax, and the expression changes of the key apoptosis-associated protein (Caspase-3 and polypeptide poly (ADP-robose) polymerase). The possible mechanism of SDT-induced apoptosis was investigated by detecting by intracellular ROS (reactive oxygen species) generation and effect of ROS scavenger-NAC (N-acetylcysteine) on SDT induced apoptosis.

Results

The intracellular PpIX increased quickly within 2 h after PpIX administration and PpIX mainly localized in the mitochondria. Compared with PpIX alone and ultrasound alone groups, the synergistic cytotoxicity of PpIX plus ultrasound was significantly boosted. In addition, the ultrasound induced some extent of chromatin condensation and MMP loss was greatly enhanced by the presence of 2 μg/ml PpIX, where PpIX alone treatment showed no or only slight effect. Time-dependent Bax translocation, caspase-3 activation and PARP cleavage were detected in SDT treatment groups. Besides, intracellular ROS production was significantly enhanced after SDT, and the general ROS scavenger NAC could obviously alleviate the SDT-caused cell viability loss, MMP loss, Bax redistribution and nuclear changes.

Conclusions

These results indicated that PpIX-mediated sonodynamic action could induce apoptosis on K562 cells, and the intracellular ROS was involved in the PpIX-SDT induced apoptosis.     

Apoptosis induced by sonodynamic treatment by protoporphyrin IX on MDA-MB-231 cells

Sonodynamic therapy (SDT) is a promising modality for cancer treatment, involving the synergistic interaction of ultrasound and some chemical compounds termed as sono-sensitizers. It has been found that SDT can lead to apoptotic cell death because of the induction of direct sonochemical and subsequent redox reactions. However, the detailed mechanisms are not clear. This study was to identify the cytotoxic effects of ultrasound-activated protoporphyrin IX (PpIX) on MDA-MB-231 cells. The fluorescence microscope was used to detect the sub-cellular localization of PpIX. Several distinct sonochemical effects were found after SDT treatment, including the decrease of cell viability, generation of intracellular ROS, the loss of mitochondrial membrane potential. The activation of some special apoptosis-associated proteins [Caspase-9, Caspase-3 and polypeptide poly (ADP-robose) polymerase] was evaluated by western blotting. The results show that PpIX mediated SDT (PpIX-SDT) treatment could obviously inhibit the proliferation of MDA-MB-231 cells, and which was significantly reduced by the pan-Caspase inhibitor z-VAD-fmk and the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC). Further, SDT induced a conspicuous loss of mitochondrial membrane potential (MMP) and a mass of ROS accumulation in MDA-MB-231 cells at 1 h post-treatment and the SDT-treated cells showed obvious Caspase-3 and Caspase-9 activation, and PARP cleavage at 6 h after treatment. And, the general apoptosis marker-Caspase-3 activation-was also greatly relieved by NAC. These findings primarily indicate a Caspase-depended apoptosis could be induced by PpIX-SDT in MDA-MB-231 cells, and the intracellular ROS was involved during the apoptotic process.     

Polydopamine Nanoparticle as a Multifunctional Nanocarrier for Combined Radiophotodynamic Therapy of Cancer

Combination of different therapeutic strategies to treat cancer has attracted tremendous attention in recent years. Herein, the authors develop polydopamine (PDA) nanoparticles with polyethylene glycol (PEG) modification as a multifunctional nanocarrier for coloading photosensitizer chlorine6 (Ce6) and curcumin (Cur) for combined photodynamic therapy (PDT) and radiotherapy (RT) of cancer. PEGylated PDA nanoparticles (PDA‐PEG) exhibit well water soluble and biocompatible in different physiological solutions and cause no obvious toxicity to cancer cells. In this nanoparticle, the loaded Ce6 can trigger the generation of single oxygen under near‐infrared laser irradiation for PDT, while the loaded Cur can act as an excellent radiosensitizer under X‐ray irradiation for enhanced external RT. As demonstrated by in vitro and in vivo therapeutic efficiency, combined PDT and RT based on PDA‐PEG/Cur/Ce6 nanoparticles exhibits significant inhibition the growth of cancer cells, revealing perfect performance in cancer treatment. Therefore, the study not only presents a polymer‐based theranostic platform for cancer treatment but also demonstrates the potential applications of combined RT and PDT for the future clinic cancer therapy.     

Apoptosis of ovarian cancer cells induced by methylene blue-mediated sonodynamic action

Objective

The present study aims to investigate apoptosis of ovarian cancer cells induced by methylene blue (MB)-mediated sonodynamic therapy (SDT).

Methods

The MB concentration was kept constant at 100 μM and ovarian cancer HO-8910 cells were exposed to ultrasound therapy for 5 s with an intensity of 0.46 W/cm². The cytotoxicity was investigated 24 h after MB-mediated sonodynamic action. Apoptosis was analyzed using a flow cytometer with Annexin V-FITC and propidium iodine (PI) staining as well as fluorescence microscopy with Hoechst 33258 staining. Intracellular reactive oxygen species (ROS) level was measured by flow cytometer with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) staining.

Results

The cytotoxicity of MB-mediated SDT on HO-8910 cells after MB-mediated SDT was significantly higher than those of other treatments including ultrasound alone, MB alone and sham treatment. Flow cytometric analysis showed a significant increase in the early and late apoptotic cell populations by MB-mediated SDT of HO-8910 cells. Nuclear condensation and increased ROS levels were also found in HO-8910 cells treated by MB-mediated SDT.

Conclusions

Our findings demonstrated that MB-mediated sonodynamic action significantly induced apoptosis of HO-8910 cells and an increase in intracellular ROS level. This indicates that apoptosis is an important mechanism of cell death induced by MB-mediated SDT. Thus, MB-mediated SDT might be a potential therapeutic strategy for combating ovarian cancer.     

Porous Lanthanum-Doped Manganese Oxide Nanoparticles for Enhanced Sonodynamic Cancer Therapy

Metal oxides hold great promise as robust sonosensitizers for sonodynamic therapy (SDT). However, they usually suffer from limited production yield of reactive oxygen species (ROS) due to the fast recombination of ultrasound-triggered electrons and holes. Herein, porous lanthanum (La)-doped MnO₂ (LMO) nanoparticles are firstly developed as promising sonosensitizers in SDT. The strategic introduction of La dopants greatly promotes the separation efficiency of ultrasound-triggered electrons and holes of MnO₂, endowing them with significantly improved ROS yield. Consequently, the LMO with polyethylene glycol decoration exhibits good SDT activity toward breast cancer cells. This work highlights the doping strategy for the development of enhanced ROS production of metal oxide sonosensitizers for SDT.     

ROS-generating alginate-coated gold nanorods as biocompatible nanosonosensitisers for effective sonodynamic therapy of cancer

Sonodynamic therapy (SDT) emerges as a promising non-invasive alternative for eradicating malignant tumours. However, its therapeutic efficacy remains limited due to the lack of sonosensitisers with high potency and biosafety. Previously, gold nanorods (AuNRs) have been extensively studied for their applications in photodynamic or photothermal cancer therapy, but their sonosensitising properties are largely unexplored. Here, we reported the applicability of alginate-coated AuNRs (AuNRs^ALG) with improved biocompatibility profiles as promising nanosonosensitisers for SDT for the first time. AuNRs^ALG were found stable under ultrasound irradiation (1.0 W/cm², 5 min) and maintained structural integrity for 3 cycles of irradiation. The exposure of the AuNRs^ALG to ultrasound irradiation (1.0 W/cm², 5 min) was shown to enhance the cavitation effect significantly and generate a 3 to 8-fold higher amount of singlet oxygen (¹O₂) than other reported commercial titanium dioxide nanosonosensitisers. AuNRs^ALG exerted dose-dependent sonotoxicity on human MDA-MB-231 breast cancer cells in vitro, with ∼ 81% cancer cell killing efficacy at a sub-nanomolar level (IC₅₀ was 0.68 nM) predominantly through apoptosis. The protein expression analysis showed significant DNA damage and downregulation of anti-apoptotic Bcl-2, suggesting AuNRs^ALG induced cell death through the mitochondrial pathway. The addition of mannitol, a reactive oxygen species (ROS) scavenger, inhibited cancer-killing effect of AuNRs^ALG-mediated SDT, further verifying that the sonotoxicity of AuNRs^ALG is driven by the production of ROS. Overall, these results highlight the potential application of AuNRs^ALG as an effective nanosonosensitising agent in clinical settings.     

Bleomycin enhances the efficacy of sonodynamic therapy using aluminum phthalocyanine disulfonate

Sonodynamic therapy (SDT), or ultrasound combined with sonosensitization, is a promising approach because it is noninvasive and penetrates deeper than light does in photodynamic therapy. We examined whether bleomycin (BLM) could improve the efficacy of SDT. We performed an in vitro study using Colon-26 cells, which are derived from mouse colon cancer. SDT with BLM was significantly more cytotoxic than SDT alone both in vitro and in vivo. We also observed an ultrasound intensity-dependent cytotoxic effect of SDT with BLM. These findings suggest that SDT with BLM might provide a novel noninvasive treatment for deep-seated tumors.     

Ultrasound exposure in the presence of hematoporphyrin induced loss of membrane integral proteins and inactivity of cell proliferation associated enzymes in sarcoma 180 cells in vitro

Ultrasonically induced effects of hematoporphyrin (HPD) on cell damage and membrane protein alteration of S180 isolated tumor cells in vitro were investigated, and the potential mechanisms of sonodynamic therapy (SDT) inhibiting tumor growth were discussed. Tumor cells suspended in air-saturated PBS (pH 7.2) were exposed to ultrasound at 1.8 MHz for up to 180 s in the presence and absence of HPD. The viability of cells was determined by a trypan blue exclusion test. To estimate the damage effects of SDT on plasma membrane of tumor cells primarily, membrane integral proteins (EGFR, Ras, Fas, FasL) and cell proliferation associated enzymes (adenylate cyclase and guanylate cyclase) were checked with immunochemical methods. The results indicated that the intensity threshold for ultrasonically induced cell damage at 1.8 MHz was 3 W/cm². At this condition, the expression of the integral proteins was obviously inhibited and the activity of the enzymes was decreased post ultrasound treatment in the presence of 20 μg/ml HPD. Loss of the membrane proteins and inactivity of AC and GC post SDT was time-dependent. This paper reveals SDT can cause the loss of tumor cell membrane integral proteins and inactivity of the enzymes associated with cell proliferation which might be attributed to a sonochemical activation mechanism. The mechanisms by that tumor growth is inhibited by SDT can be understood as that the growth signaling pathway is partially interdicted and the resistance of tumor cells to the specifically activated immune cells is weakened.     

Oxygen and Indocyanine Green loaded microparticles for dual-mode imaging and sonodynamic treatment of cancer cells

Oxygen and Indocyanine Green (ICG) loaded microparticles (OI-MPs) were fabricated by a gas-driven coaxial flow focusing (CFF) process for dual-mode imaging and sonodynamic therapy (SDT). The produced OI-MPs agent showed stable optical properties, superior imaging depth in near infrared (NIR) fluorescence imaging, and enhanced acoustic contrast after ultrasound mediation. We hypothesized that encapsulating ICG and oxygen in microparticles would enhance reactive oxygen species (ROS) production in SDT. This hypothesis was validated in a cell-free environment. We further hypothesized that ultrasound mediated fragmentation of the OI-MPs would induce cytotoxicity and apoptosis of cancer cells. This hypothesis was validated in SKOV3 ovarian cancer cells. Our research demonstrated that OI-MPs can be potentially used as a dual-mode theranostic agent for image guided SDT with enhanced efficacy. Further study is needed to delineate the mechanism of ROS-induced cell apoptosis and optimize the OI-MPs formulation for the maximal anti-cancer potency.     

Enhanced sonodynamic therapy by carbon dots-shelled microbubbles with focused ultrasound

Sonodynamic therapy involving the non-invasive and local generation of lethal reactive oxygen species (ROS) via ultrasound (US) with sonosensitizers has been proposed as an emerging tumor therapy strategy. However, such therapy is usually associated with inertial cavitation and unnecessary damage to healthy tissue because current sonosensitizers have insufficient sensitivity to US. Here, we report the use of a new proposed sonosensitizer, carbon dots (C-dots), to assemble microbubbles with a gas core (C-dots MBs). As the C-dots were directly integrated into the MB shell, they could effectively absorb the energy of inertial cavitation and transfer it to ROS. Our results revealed the appearance of ¹O₂, •OH, and H₂O₂ after US irradiation of C-dots MBs. In in vitro experiments, treatment with C-dots MBs plus US induced lipid peroxidation, elevation of intracellular ROS, and apoptosis in 32.5%, 45.3%, and 50.1% of cells respectively. In an animal solid tumor model, treatment with C-dots MBs plus US resulted in a 3-fold and 2.5-fold increase in the proportion of ROS-damaged cells and apoptotic cells, respectively, compared to C-dots MBs alone. These results will pave the way for the design of novel multifunctional sonosensitizers for SDT tumor therapy.     

Hypocrellin B-mediated sonodynamic action induces apoptosis of hepatocellular carcinoma cells

Objective

The present study aims to investigate apoptosis of hepatocellular carcinoma cells induced by hypocrellin B-mediated sonodynamic action.

Methods

The hypocrellin B concentration was kept constant at 2.5 μM and cells from the hepatocellular carcinoma HepG2 cell line were exposed to ultrasound with an intensity of 0.46 W/cm² for 8 s. Cell cytotoxicity was quantified using an MTT assay 24 h after sonodynamic therapy (SDT) of hypocrellin B. Apoptosis was investigated using a flow cytometry with Annexin V-FITC and propidium iodine staining. Intracellular reactive oxygen species (ROS) levels were detected using a flow cytometry with 2,7-dichlorodihydrofluorecein diacetate (DCFH-DA) staining.

Results

The cytotoxicity of hypocrellin B-mediated sonodynamic action on HepG2 cells was significantly higher than those of other treatments including ultrasound alone, hypocrellin B alone and sham treatment. Flow cytometry showed that hypocrellin B-induced sonodynamic action markedly enhanced the apoptotic rate of HepG2 cells. Increased ROS was observed in HepG2 cells after being treated with hypocrellin B-mediated sonodynamic action.

Conclusions

Our data demonstrated that hypocrellin B-mediated sonodynamic action remarkably induced apoptosis of HepG2 cells, suggesting that apoptosis is an important mechanism of cell death induced by hypocrellin B-mediated SDT.     

Detection and comparison of reactive oxygen species (ROS) generated by chlorophyllin metal (Fe, Mg and Cu) complexes under ultrasonic and visible-light irradiation

In this paper, in order to examine the mechanisms of sonodynamic and photodynamic reactions, the chlorophyllin metal (Chl-M (M=Fe, Mg and Cu)) complexes were irradiated by ultrasound (US) and visible-light (VL), respectively, and the generation of reactive oxygen species (ROS) were detected by the method of Oxidation-Extraction Spectrometry (OES). That is, the 1,5-diphenyl carbazide (DPCI) is oxidized by the generated ROS into 1,5-diphenyl carbazone (DPCO), which can display a various visible absorption around 563 nm wavelength. Besides, some influence parameters on the generation of ROS were also reviewed. The results demonstrated an apparent synergistic effect of Chl-M and ultrasonic or visible-light irradiation for the generation of ROS. Moreover, the quantities of generated ROS increase with the increase of (ultrasonic or visible-light) irradiation time and Chl-M (M=Fe, Mg and Cu) concentration. Finally, several quenchers were used to determine the kind of the generated ROS. It is wished that this paper might offer some valuable references for the study on the sonodynamic therapy (SDT) and photodynamic therapy (PDT) mechanisms and the application of Chl-M in tumor treatment.     

Sonodynamic action of pyropheophorbide-a methyl ester induces mitochondrial damage in liver cancer cells

Sonodynamic therapy with pyropheophorbide-a methyl ester (MPPa) presents a promising aspect in treating liver cancer. The present study aims to investigate the mitochondrial damage of liver cancer cells induced by MPPa-mediated sonodynamic action. Mouse hepatoma cell line H₂₂ cells were incubated with MPPa (2 μM) for 20 h and then exposed to ultrasound with an intensity of 0.97 W/cm² for 8 s. Cytotoxicity was investigated 24 h after sonodynamic action using MTT assay and light microscopy. Mitochondrial membrane potential (ΔΨm) was analyzed using flow cytometry with rhodamine 123 staining and ultrastructural changes were observed using transmission electron microscopy (TEM).The cytotoxicity of MPPa-mediated SDT on H₂₂ cell line was 73.00 ± 3.42%, greater than ultrasound treatment alone (28.12 ± 5.19%) significantly while MPPa treatment alone had no significant effect on H₂₂ cells. Moreover, after MPPa-mediated SDT cancer cells showed swollen mitochondria under TEM and a significant collapse of mitochondrial membrane potential. Our findings demonstrated that MPPa-mediated SDT could remarkably induce cell death of H₂₂ cells, and highlighted that mitochondrial damage might be an important cause of cell death induced by MPPa-mediated SDT.     

Size and Shape Effects of Near-Infrared Light-Activatable Cu2(OH)PO4 Nanostructures on Phototherapeutic Destruction of Drug-Resistant Hypoxia Tumors

Development of simple, robust, and noninvasive therapeutic approaches to treat cancers and improve survival rates is a grand challenge in clinical biomedicine. In particular, the sizes and shape of the nanomaterials play a vital role in dictating their biodistribution and clearance pathways. It remains elusive how the size and shape of a nanomaterial affect its therapeutic efficacy in cancer diagnosis and treatments. To tackle the above problem, the effects of size and shape of Cu₂(OH)PO₄ nanostructures (nanosheets and quantum dots) on the photodynamic therapy (PDT) in destroying malignant drug-resistant lung tumors and on combating the tumor hypoxia problem are investigated and compared. The photocatalytic mechanism of Cu₂(OH)PO₄ nanostructures mainly involves the generation of reactive oxygen species (ROS), such as hydroxyl radical (^·OH) and singlet oxygen (¹O₂). Under an oxygen deprivation condition, Cu₂(OH)PO₄ nanosheets still can generate OH radicals to kill cancer cells upon near-infrared (NIR) light irradiation. Overall, in vitro and in vivo experiments show that Cu₂(OH)PO₄ nanosheets can overcome tumor hypoxia problems and effectively mediate dual modal PDT and photothermal therapeutic (PTT) effects on destruction of NCI-H23 lung tumors in mice using ultralow doses (350 mW cm⁻²) of NIR (915 nm) light.     

Combination of sonodynamic with temozolomide inhibits C6 glioma migration and promotes mitochondrial pathway apoptosis via suppressing NHE-1 expression

Temozolomide (TMZ) was used for clinical postoperative or non-surgical chemotherapy patients. However, its effect remains unsatisfactory and gradually discovered that the presence of chemoresistance. To explore more effective therapy using TMZ, we investigate the effects of combination of application of TMZ together with Sonodynamic therapy (SDT), which is based on the ultrasonic activation of a sonosensitizer, with low toxicity, noninvasive, deeper penetrability and a promising approach for treating malignant glioma by inducing apoptosis on glioma cells in vitro. Sodium–hydrogen exchanger isoform 1 (NHE1), which enable glioblastoma cells to escape TMZ-mediated toxicity via increased H⁺ extrusion and affect the apoptosis effect on C6 glioma cells in vitro. The C6 cells survival rate and time point of TMZ resistance were tested by the Cell Counting Kit-8 (CCK8) viability assay. Western blot analysis results showed that the expression of NHE1 and matrix metalloproteinase-2 (MMP-2) protein obviously decreased by TMZ + SDT. Meanwhile, combined treatments enhanced the expression of mitochondrial pathway apoptosis proteins, as well as suppressed MMP-2 to weaken the migration ability in TMZ-resistant C6 cell line. These results provided the first evidence that the sensitivity of TMZ chemotherapy in resistant malignant glioma may be improved by SDT.     

Sonodynamically induced anti-tumor effect with protoporphyrin IX on hepatoma-22 solid tumor

Objective

The purpose of this study was to evaluate sonodynamically induced anti-tumor effect of protoporphyrin IX (PPIX) in mice bearing hepatoma-22 (H-22) solid tumors, and the possible in vivo cell damage mechanism was also investigated.

Methods

The pharmacokinetics of PPIX was analyzed in plasma, skin, muscle and tumor of H-22 bearing mice. Tumors were irradiated with ultrasound (1.43 MHz, I_SATA 3 W/cm², 3 min) for three times at 8, 12 and 24 h after 5.0 mg/kg PPIX administration, respectively. The anti-tumor effects of sonodynamic therapy (SDT) were estimated by the tumor inhibition ratio (volume and weight). The bio-effects of SDT were evaluated by hematoxylin and eosin (H&E) staining, transmission electron microscope (TEM) observation, lipid peroxidation (LPO) measurement and anti-oxidative enzymes (glutathione peroxidase (GSH-PX), catalase (CAT) and superoxide dismutase (SOD)) assay.

Results

A significant anti-tumor effect was obtained by PPIX-mediated sonodynamic therapy (PPIX-SDT). At the fifteenth day after PPIX-SDT, the tumor growth and tumor weight inhibition ratios were 53.84% and 45.86%, respectively. In addition, the structure of tumor tissues and the anti-oxidative enzymes were obviously destroyed after SDT treatment.

Conclusions

A biochemical mechanism was involved in PPIX-SDT in vivo, and the free radicals produced by the synergistic treatment destroying the anti-oxidative system of tumor cells in vivo may play an important role in this action. Also, the thermal effect could not be excluded in inducing damage of cellular structures, like membrane disruption and chromatin condensation under current evaluation in this paper.     

Modulating Endogenous Oxygen Consumption Enhanced AIEgens-Mediated Photodynamic Therapy against Advanced Bladder Tumor

Photodynamic therapy (PDT) plays an important role in tumor therapy. However, PDT outcomes remain poor due to the insufficient reactive oxygen species (ROS) yield of the photosensitizer and the aggravation of hypoxia in the tumor microenvironment through PDT. To solve this issue, a puissant PDT nanoparticles (NPs) platform is developed to defeat tumors by employing powerful PDT in an oxygen-enriched tumor microenvironment and by inhibiting tumor migration through regulating hypoxia-related pathways. Specifically, TFM, as a previously reported AIEgen, does not emit brightly but instead makes use of its nonradiative relaxation for ROS generation, along with oxygen regulator doxycycline (DOXY), both of which are encapsulated in NPs that can specifically penetrate deep into tumor tissue. After released from the NPs, TFM functions as a powerful ROS generator, and DOXY reduces endogenous oxygen consumption via regulating mitochondrial cytochrome oxidase 1, further enhancing TFM-mediated PDT efficacy. Meanwhile, reoxygenation is observed to reduce the expression of hypoxia inducible factor-1α and M2 macrophages induced by PDT. The amplified PDT not only strongly killed tumor but, along with the downregulated hypoxia related pathways during the post-PDT process, inhibited tumor migration. In summary, such NPs exhibit potent anti-tumor effects for advanced bladder cancer therapy.