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Although cancer has been the main reason for death for decades, until now, no safe certified drugs have been discovered to efficiently inhibit its progression. Currently, we aim to employ an in-silico model to predict the action mechanism of phenolic components that we previously demonstrated as inhibitors of colon, prostate and lung cancer. The work was performed by testing pharmacokinetics and pharmacodynamics proprieties of selected molecules. Four compounds: Kaempferol, chrysin, vanillin and p-hydroxybenzoic acid widely obeyed the five rules and the ADME/T criteria with minimum toxicity. Docking prediction recorded an intense affinity between kaempferol-CDK1(−17.72 ± 0.02 kcal/mol), chrysin-CDK1 (−17.61 ± 0.01 kcal/mol) and kaempferol-caspase 8 (−9.30 ± 0.00 kcal/mol), chrysin-caspase 8 (−8.18 ± 0.12 kcal/mol), vanillin-MMP9 (−9.83 ± 0.04 kcal/mol) and p-hydroxybenzoic acid-Ca2 (−8.91 ± 0.01 kcal/mol). Likewise, MMGBSA results demonstrated that these compounds are evidently bound to the targets’ active sites with solid interactions. The passed compounds might target the responsible factors for transcription as inactivators. The molecular mechanism based on kaempferol and vanillin actions was predicted to target CDK1 and MMP9 via cell cycle arrest and apoptosis. All results were confirmed through RMSD analysis suggesting that kaempferol and vanillin are the best ligands to suppress cancer cell proliferation. Thus, these findings allowed us to imagine a potential molecular mechanism of these binaries, phenolic compounds/target proteins to lead further trails on cancer, of which the synergistic activity between phenolic compounds could promote the therapeutic capacity.

Aims: This study aims to prepare and investigate the potential of theobromine derivative-loaded chitosomes as an effective anticancer drug. Background: $N$-Benzyl-2-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1$H$-purin-1-yl)acetamide (T-1-BA) exhibited promising anticancer potential. Loaded chitosomes, highlighting their suitability as a drug delivery system. The study builds on the significance of targeted drug delivery systems for enhanced anticancer efficacy. Objectives: To prepare T-1-BA-loaded chitosomes (T-1-BA-PC-CS complex) using the thin film hydration technique. To examine the colloidal characteristics, entrapment efficiency, and stability of the T-1-BA-PC-CS complex. To assess the in vitro anticancer efficacy of the T-1-BA-PC-CS complex against Hct116 and A549 cancer cell lines comparing the free T-1-BA, blank chitosomes, and the standard EGFR inhibitor, Lapatinib. Methods: The T-1-BA-PC-CS complex was prepared through the thin film hydration technique. Colloidal characteristics, including particle size, PDI, and zeta potential, were examined. In vitro anticancer efficacy was assessed through IC${}_{50}$ values, comparative analyses, SI calculations, cell cycle analysis, flow cytometry, and wound healing assays. Results: The T-1-BA-PC-CS complex demonstrated reduced IC${}_{50}$ values against Hct116 and A549 cell lines, indicating enhanced cytotoxicity compared to free T-1-BA and blank chitosomes. Comparative analyses highlighted superior anticancer activity. SI values indicated preferential cytotoxic effects on cancer cell lines. Flow cytometry analysis revealed cell cycle alterations and increased apoptosis. Wound healing assays showed a significant impact on migration and wound healing in A549 cells. Conclusion: The findings suggest that the T-1-BA-PC-CS complex holds great promise as an anticancer therapeutic by efficiently inducing favorable alterations in cell cycle phases and apoptosis, demonstrating its potential for further development in cancer treatment.

Photodynamic therapy is a binary treatment now accepted in clinic for various malignancies in several countries around the world. Phthalocyanine molecules are second-generation photosensitizers with enhanced photophysical and photochemical properties over those of porphyrins. They have been shown to be phototoxic against a number of cell types and tumor models. A great deal of research has been devoted to the elucidation of their mechanism of action and mode of cell death. The present paper reviews phthalocyanine pre-clinical anti-cancer research with emphasis on phthalocyanine induced apoptosis using a silicon phthalocyanine, Pc4. A brief summary of the latest clinical results using phthalocyanines is presented.

The cytotoxic response to photodynamic therapy can involve apoptosis, necrosis or both. Using agents with known patterns of sub-cellular localization, we assessed different sites of photodamage as a determinant of cell death, using murine leukemia cells in vitro. Mitochondrial or mitochondrial + lysosomal photodamage led to a rapid apoptotic response, associated with the release of cytochrome c from mitochondria into the cytosol. This occurred immediately after irradiation of photosensitized cells. When photodamaged cells were warmed to 37 °C, there was a rapid apoptotic response. Lysosomal photodamage led to the immediate release of cathepsins and other proteolytic enzymes. During a subsequent incubation at 37 °C, there was a slow loss of the mitochondrial membrane potential, with cytochrome c appearing in the cytosol within 30 min. These effects derive from proteolytic effects of lysosomal enzymes on mitochondria. The apoptotic response to lysosomal photodamage was both slow and incomplete, with many non-viable cells not exhibiting apoptotic morphology. The latter result was correlated with photodamage to procaspase-3, an effect not observed when mitochondria were the predominant target for photodamage. Depending on the sub-cellular target, photodynamic therapy can either activate or inhibit critical elements of apoptosis.

We report two medical applications of particle-induced X-ray emission (PIXE) as described below (1) Observation of biological events: The kinetics of trace elements during the initiation of radiation-induced apoptosis (RIA) was observed using a micro-PIXE and PIXE. RIA is a process in which irradiated cells commit suicide; it results in the removal of severely damaged and harmful cells. During RIA, cytochrome c is released from the mitochondria and reaches the nucleus, where it activates a Ca- or Mg-dependent endonuclease. We examined this phenomenon by using a micro-PIXE and PIXE. A high concentration of Fe was detected in the stroma of cells in the early apoptotic phase. We also observed accumulation of large amounts of Ca and Mg in the nucleus.

(2) Development of liquid-core microcapsules for novel cancer chemoradiotherapy: Currently, we are developing liquid-core (containing an anticancer drug) microcapsules that release their core content upon irradiation. These microcapsules will localize the anticancer drug within the irradiated field. The outer shell of these microcapsules is prepared from alginate and hyaluronic acid and polymerized by Fe, while the anticancer drug Paraplatin^® (carboplatin) containing Pt is the liquid core. The micro-PIXE revealed that these microcapsules released their core content after irradiation, and the amount of carboplatin released was measured by PIXE. More than 83.1% ± 8.3% of the microcapsules were ruptured, and the amount of carboplatin released was more than 81.2% ± 2.3%. Thus, the combination of radiotherapy and chemotherapy showed improved antitumor effects and a decrease in adverse effects because of drug localization.

The abnormal cell exit of apoptosis from S-phase, correlation between apoptosis and Mg, and between its S-phase fraction and Zn were studied in inoculated Sarcoma-180 receiving 10 or 20 Gy of ⁶⁰Co gamma ray, IN VIVO, in BALB/c mice.

The apoptosis and S-phase fraction were detected by double immunostaining: 1) TUNEL that colors apoptosis brown; and 2) histone H3 mRNA probe that results in blue deposition to S-phase of cells. The concentrations of Mg and Zn were studied by PIXE.

The Mg strongly correlated with the frequency of apoptosis, but other kinds of cells did not. On the contrary, The S-phase fraction of apoptosis did not correlated with Zn, but S-phase of other kinds of cells did. This different correlation suggests the different metabolism between apoptosis and other kinds of cell.

The interactions between mitochondrial and nuclear Mg concentration for the development of radiation-induced apoptosis were tested IN VIVO in Sarcoma-180 in BALB/c mice.

The frequency of apoptosis was expressed as a percentage of TUNEL reactivity in the 5 microscopic views under 400 times magnification, and Mg concentration in either mitochondria or nucleus was measured by Particle Induced X-ray Emission (PIXE), on 3, 6, 9, 12 and 24 hours after the 10 or 20 Gy of ⁶⁰Co gamma ray radiation, to the mice with or without the 0.01 mMOL MgCl₂ oral administration.

There were proximal increases of apoptosis, which peaked on 9 hours after radiation. The Mg concentration of mitochondria strongly correlated with frequency of apoptosis from 3 to 9 hrs after radiation, and that of the nucleus strongly correlated with frequency of apoptosis from 6 to 24 hours after radiation. The oral administration of MgCl₂ did not change those correlations.

The two steps are considered: 1st) signaling from mitochondria to nucleus involving Mg on induction of apoptosis; and 2nd) removal of apoptosis involving nuclear Mg.

We evaluated the effect of Bu-Zhong-Yi-Qi-Tang, a prescription of traditional Oriental medicine, and its major ingredients on protection of the intestine and hematopoietic organs against radiation damage in this study. The jejunal crypt survival, endogenous spleen colony formation, and apoptosis in jejunal crypt cells were investigated in mice irradiated with high and low doses of γ-rays. Bu-Zhong-Yi-Qi-Tang administration before irradiation protected the jejunal crypts (p < 0.0001), increased the formation of the endogenous spleen colony (p < 0.05) and reduced the frequency of radiation-induced apoptosis (p < 0.05). In experiments on the effects of the individual ingredient of Bu-Zhong-Yi-Qi-Tang, Rensan (Radix Ginseng), Danggui (Radix Angelicae gigantis), Shengma (Rhizoma Cimicifugae) and Chaihu (Radix Bupleuri) might have major radioprotective effects, and each might have different degrees of effect on these three endpoints. These results indicated that Bu-Zhong-Yi-Qi-Tang might be a better agent than any one of its ingredients to satisfy all three endpoints. Although the mechanisms of this inhibitory effect remain to be elucidated, these results indicated that Bu-Zhong-Yi-Qi-Tang might be a useful radioprotector, especially since it is a relatively non-toxic natural product. Further studies are needed to better characterize the protective nature of Bu-Zhong-Yi-Qi-Tang extract and its ingredients.

In this experiment, we investigate the biochemical effects of traditional Chinese medicines via an in vitro bone cell culture. Ten different Chinese medicines were used in this study. The rat osteoblast-osteoclast co-culture system was used as the experimental model. After the cells grew to 80% confluence, various tested materials were added. The mitochondria activity of the bone cells after exposure to various preparations of Chinese medicines was determined by colorimetric assay. Biochemical markers such as protein content, lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and acid phosphatase (ACP) titer were analyzed to evaluate the bone cell activity. When cultured with various Chinese medicines for 24 hours, only four of these ten Chinese medicines had potential beneficial effects on the bone cell culture; and only Drynaria fortunei (Kunze) J. Sm. had a universal beneficial effect on bone cell metabolism. The major beneficial effect of Drynaria fortunei (Kunze) J. Sm. on bone cells is probably mediated by the induction of apoptosis of the osteoclast cell population. Continued study of alterations in gene expression of bone cells caused by Chinese medicines will improve our understanding of bone cell responses to various pharmacological interventions.

Oxidative stress has been implicated in the pathogenesis of different neurodegenerative disorders. To investigate the protective effects of Wuyaoshunqisan against H₂O₂-induced apoptosis in the central nervous system, the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) method, flow cytometric analysis, and the DNA fragmentation assay were performed on cells of the hippocampal cell line HiB5. Through the morphological and biochemical analyses, it was shown that HiB5 cells treated with H₂O₂ exhibit classical apoptotic features, while the occurrence of such changes is reduced in cells pre-treated with Wuyaoshunqisan prior to H₂O₂ exposure.

The protective effect of Acanthopanax senticosus (AS) against ethanol (EtOH)-induced apoptosis of the human neuroblastoma cell line SK-N-MC was investigated via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometric analysis, DNA fragmentation assay, reverse transcription-polymerase chain reaction (RT-PCR), and caspase-3 assay. It was shown that cells treated with EtOH exhibit classical apoptotic features, while cells pre-treated with Acanthopanax senticosus prior to EtOH exposure showed decreased occurrence of apoptotic features. In addition, Acanthopanax senticosus pre-treatment was shown to inhibit EtOH-induced increase in caspase-3 mRNA expression and activity. These results suggest that Acanthopanax senticosus may exert a protective effect against EtOH-induced apoptosis of human neuroblastoma cells.

The effects of Saikosaponin-A on human breast cancer cell lines (MDA-MB-231 and MCF-7) were investigated. Results demonstrated that Saikosaponin-A inhibited the proliferation or viability of the MDA-MB-231 and MCF-7 cells in a dose-dependent manner. Saikosaponin-A treatment of MDA-MB-231 for 3 hours and of MCF-7 cells for 2 hours, respectively caused an obvious increase in the sub-G1 population of cell cycles. Apoptosis in MDA-MB-231 cells was independent of the P53/p21 pathway mechanism and was accompanied by an increased ratio of Bax to Bcl-2 and c-myc levels and activation of caspase-3. In contrast, apoptosis of MCF-7 cells may have been initiated by the Bcl-2 family of proteins and involved p53/p21 dependent pathway mechanism, and was accompanied by an increased level of c-myc protein. Both the apoptosis of MDA-MB-231 cells and MCF-7 cells showed a difference worthy of further research.

In order to develop a new apoptosis inducer, we screened 22 crude drugs for their apoptosis-inducing activity. It was found that Glycyrrhiza uralensis, Cynomorium songaricum, Eucommia ulmoides, Phellodendron amurense, Cinnamomum cassia and Paeonia lactiflora induced the death of HL-60 cells. To investigate the mechanism of apoptosis induced by these six crude drugs, the mitochondrial transmembrane potential and the activity of caspase-3 were measured. Reduced mitochondrial transmembrane potentials within 12 hours after the administration of Glycyrrhiza uralensis, Cynomorium songaricum, Phellodendron amurense and Paeonia lactiflora, and within 24 hours after the administration of Eucommia ulmoides and Cinnamomum cassia were observed. All of the six apoptosis-inducing crude drugs increased caspase-3 activity within 12–36 hours after administration. After further examining the apoptosis-inducing activity of berberine, palmatine, panelofuroline and glycyrrhizin, which were the ingredients obtained from Phellodendron amurense, Glycyrrhiza uralensis and Paeonia lactiflora, it was found that only berberine could induce apoptosis. From these results, it was concluded that the apoptosis induced by the six crude drugs (Glycyrrhiza uralensis, Cynomorium songaricum, Eucommia ulmoides, Phellodendron amurense, Cinnamomum cassia and Paeonia lactiflora) occurred via the mitochondrial route and that the apoptosis-conducting mechanism acted through a cascade involving caspase-3.

Lindera strychifolia, a scandent shrub Lauraceous medicinal plant, has been used in Chinese traditional medicine as a palliative and an anti-spasmodic. It also shows cytotoxic effects against several tumor cell lines and inhibits marcromolecule biosynthesis. This study investigated the anti-tumor effects of L. strychifolia extract against lung cancer cells using in vitro and in vivo models. Two human lung cancer cell lines A549 (adenocarcinoma) and SBC-3 (small cell carcinoma), and a non-tumor cell line 3T3-L1 (mice fibroblasts) were subjected to L. strychifolia extract treatment. On lung cancer cells, L. strychifolia induced cell growth inhibition in a dose-dependent manner. Conversely, the extract did not show any significant cytotoxic effect on 3T3-L1 cells. Therefore, the extract is specific for tumor cells. Tumor cells treated with L. strychifolia extract showed typical morphological appearance of apoptosis including nuclei fragmentation and cell condensation. The in vivo effects of L. strychifolia extract were investigated in C57BL/6 mice transplanted with Lewis lung cancer (LL-2) cells, and in BALB/c nude mice transplanted with A549 or SBC-3 human lung cancer cells. Oral administration of L. strychifolia extract prolonged survival time and inhibited tumor growth in a dose-dependent manner by inducing apoptosis in the LL-2 cell mice model. Furthermore, in A549 or SBC-3 cell nude mice models, oral administration of L. strychifolia extract also significantly inhibited tumor growth at the 5.0 mg/ml concentration. These findings suggested that the components of L. strychifolia have anticancer activity and may contribute to clinical applications in the prevention and treatment of lung cancer.

Cell apoptosis is now known to play an important role in the maintenance of cellular homeostasis and anti-carcinogenesis. The anticancer effect of aqueous extract prepared from Phyllanthus urinaria (P. urinaria) was investigated by analyzing its potential to induce apoptosis in human cancer cells. We showed that the aqueous extract of P. urinaria could reduce the viability by inducing the apoptosis in human cancer cells derived from several different origins as demonstrated by morphological changes and DNA fragmentation. Yet, P. urinaria extract exhibited no cytotoxic effect on normal human cells, including vascular endothelial cells and liver cells under the same conditions. It suggests that the aqueous extract of P. urinaria is substantially useful in treating various kinds of human cancer cells without toxic side effect on normal cells.

Hepatocellular carcinoma is an important health problem in Asia. A blend of herbal extracts containing radix bupleuri (KY88) was tested for its effects on liver cancer cells. A hepatocellular carcinoma cell line (HB8064) was cultured with methanol extract of KY88. We were able to produce a dose-dependent inhibition of cancer cell proliferation. At IC₅₀ and IC₁₀₀, KY88 induces a DNA ladder pattern, indicating the presence of apoptosis. We also checked the changes of the levels of interleukin (IL)-2, -4 and -6, interferon (INF)-γ and tumor necrosis factor (TNF)-α by ELISA kits. After 24 hours of culture, there was activation of IL-2 and -4 and TNF-α. However, significant changes were observed only for IL-4 and TNF-α. Therefore, we concluded that KY88 is able to induce apoptosis, which may be regulated through changes in IL-4 and TNF-α.

We studied the effect of 4-acetyl-12,13-epoxyl-9-trichothecene-3,15-diol (AETD) isolated from Isaria japonica, one of the most popular Chinese fungal medicines, on the induction of apoptosis in rat bladder carcinoma NBT-II cells. AETD was cytotoxic to NBT-II cells, and this cytotoxic effect appears to be attributed to its induction of apoptotic cell death, as AETD induced nuclear morphological changes and internucleosomal DNA fragmentation, and increased the proportion of hypodiploid cells and activity of caspase-3. AETD treatment also decreased the expression of the anti-apoptotic protein Bcl-2 and increased the expression of the pro-apoptotic protein Bax. These results provide important information in understanding the mechanism(s) of AETD-induced apoptosis.

The protective effects of two Chinese herbs, Astragalus mongholicus, Polygonum multiflorum and Astragalus mongholicus-Polygonum multiflorum in combination against thymus injury induced by cyclophosphamide were evaluated by transmission electron microscopy, image analysis, DNA gel electrophoresis as well as flow cytometry. Results showed that mice pretreated with cyclophosphamide had degenerated thymus with less normal thymocytes; when those mice were treated with the herbs, thymus morphology improved. The apoptosis analysis showed the thymus treated with the herbs had fewer apoptotic thymocytes than the thymus pretreated with cyclophosphamide only. In conclusion, Astragalus mongholicus and Polygonum multiflorum have protective effects on the thymus against cyclophosphamide-induced injury. Their protective effects partly attribute to reduced apoptosis. Astragalus mongholicus-Polygonum multiflorum in combination has better effects than either of the two herbs.

Coptidis rhizoma has been used as traditional herb medicine in gastrointestinal disorders in the Eastern Asia. We investigated whether the anticancer effects of the C. rhizoma induced apoptosis on human colorectal cancer cells SNU-C4. The cytotoxic effect of C. rhizoma was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. To determine apoptotic cell death, 4,6-diamidino-2-phenylindole (DAPI) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, reverse transcription-polymerase chain reaction (RT-PCR) and caspase-3 enzyme assay were performed. In this study, C. rhizoma treatment (100 μg/ml) revealed typical morphological apoptotic features. Additionally, C. rhizoma treatment (100 μg/ml) increased levels of BAX and CASPASE-3, and decreased levels of BCL-2. Caspase-3 enzyme activity by treatment of C. rhizoma (100 μg/ml) also significantly increased compared to the control (p<0.05). These data indicate that C. rhizoma caused cell death by apoptosis through caspase pathways on human colorectal cancer cells SNU-C4.

Chingwaysan, a Chinese herbal formula, contains Cimicfugae Rhizoma, Rehmanniae Radixet Rhizoma, Moutan Radicis Cortex, Coptidis Rhizoma and Angelicae Sinensis Radix. This medicine is well-known for its curing power for ulcerated gums, toothaches, cheek boils and bleeding gingiva. However, no reports can be found on its application in the treatment of oral cancers. We are therefore interested in whether Chingwaysan is capable of causing abnormal apoptosis processes, and whether this condition can be rectified through Chingwaysan herb treatment. We used aqueous extract to treat OC2 and TSCCa cells (both are human oral cancer cell lines) with different Chingwaysan concentrations (0, 10, 25, 50, 75 and 100 μl/ml). The MTT (3, (4, 5-dimethyl-thiazol) 2, 5-diphenyl-tetraxolium bromide) reduction assay was employed to quantify the differences in cell activity and viability. DNA ladder formation on agarose electrophoresis was also performed. The bax expression level was monitored using immunoblotting techniques. The patterns of the changes in expression were scanned and analyzed by NIH image 1.56 software. Taken together, drastic morphological changes, reduced cell viability and the presence of inter-nucleosomal DNA fragmentation all indicated that Chingwaysan is capable of inducing apoptosis in OC2 and TSCCa cell lines. Furthermore, the accumulation of wild type bax protein significantly increased in a dose-dependent manner upon treatment with Chingwaysan. In conclusion, Chingwaysan can induce apoptosis via a bax-dependent pathway in cells from these two particular oral cancer cell lines.