Synergistic effect of sorafenib with Platycladus orientalis (L) leaf extract on cervical cancer

The extracts of Platycladus orientalis (L.) Franco leaves have shown promising anti-cancer, anti-oxidant and anti-inflammatory potency with the traditional knowledge of healing HPV associated warts. The purpose of this research is to assess the synergistic activity of sorafenib and Platycladus orientalis (L) leaf extraction on cervical cancer cells. The cytotoxicity efficiency of different concentrations of Sorafenib and ethanol extract of Platycladus orientalis (L.) leaves were tested on HeLa cells by MTT and Trypan blue assays. The synergistic effect of the IC50 concentrations of Sorafenib and Platycladus orientalis (L.) on HeLa cell by MTT assay, and mRNA expression levels of tumor suppressor tazarotene-induced gene 3 (TIG3), proliferating cell nuclear antigen (PCNA) gene and apoptosis modulator (Bcl-2) gene by RT-PCR were evaluated with individual treatments. Combination treatment showed a relatively more expression of TIG3 and less expression of Bcl-2 and PCNA was observed. Growth factor-induced MAPKP activation was arrested by compound combination treatment, which and suppression of proliferation-induced apoptosis of cervical cancer cells. Based on the our results, the combination of sorafenib and crude leaf extract from Platycladus orientalis (L.) can effectively suppress cervical cancer cell growth, thereby providing an interesting rationale for further clinical trials and in-vivo studies.

Regulation of inflammation and COX-2 gene expression in benzo (a) pyrene induced lung carcinogenesis in mice by all trans retinoic acid (ATRA).

AIM: Inflammation provides favourable microenvironment for cancer development. An enhanced COX-2 gene expression is a key inflammatory mediator of cancers and the drug that inhibits it, helps to manage cancer effectively and increases survival rate. The objective is to analyse the inflammatory changes and COX-2 gene expression in benzo (a) pyrene induced mice and to evaluate the regulatory effect of all trans retinoic acid. MATERIALS AND METHODS: The body and organ weights were recorded in B(a)P induced mice. The haematological parameters and serum inflammatory markers of carcinogenesis were tested. The H & E stained liver and lung tissues were examined for histopathologic changes. The COX-2 gene expression was analysed by RT-PCR and qPCR in lung and liver. KEY FINDINGS: The decreased body weight, increased organ weights and the damages in liver and lung were observed in B(a)P induced mice and were prevented significantly upon ATRA treatment. The lowered Hb, RBC and lymphocytes and an enhanced WBC, monocytes and neutrophils observed in B(a)P group were significantly reversed in treated group. A drastic increase in cancer associated inflammatory markers observed in B(a)P induced mice were significantly (P ≤ 0.001) reduced in treated mice. The RT-PCR product density of COX-2 gene was very high in B(a)P group (lung-0.43 ± 0.06; liver-0.39 ± 0.04) significantly lower in treated group (lung-0.12 ± 0.03; liver-0.08 ± 0.03) with a significant difference in RQ values (B(a)P lung-18.46 ± 0.04, liver-12.46 ± 0.08; treated lung-5.93 ± 0.07, liver-2.92 ± 0.10). SIGNIFICANCE: The ATRA has decreased the inflammatory condition with downregulation of COX-2 gene expression and thereby prevented carcinogenesis during early stage of B(a)P induced cancer development.

Protective role of All Trans Retinoic Acid on B16F10 melanoma cell line metastasis in C57BL/6 mice by enhancing RAR- ß protein and homeostasis maintenance.

Lung cancer is the leading cancer according to the World Health Organization (WHO), resulting in highest death rate worldwide due to the high level of metastasis. Hence, the drugs that protect from metastasis either as an adjuvant or a primary therapeutic agent may help to reduce the death rate. In this study, All Trans Retinoic Acid (ATRA) was tested for its action against metastatic lodging of B16F10 melanoma cells in the lung and liver of the C57BL/6 mouse model. Serum, lung and liver were evaluated biochemically for the cancer associated changes. Metastatic cancer development was confirmed by tumor nodule formation and histopathological analysis. RAR-ß protein expression was analyzed by immunohistochemistry and histopathology. ATRA treated mice showed a percentage of inhibition on metastatic tumor growth in lung and liver and a corresponding protection against pathological changes in these organs. Cholesterol and γ-Glutamyl Transferase (GGT) levels found in cancer induced mice were reduced in the ATRA treated group. As compared to the normal group, lung tissue from cell line induced cancer control group had less RAR-ß protein expression while the ATRA treated group showed enhanced RAR-ß protein expression. This indicates that the anti-metastasis effects of ATRA might have shown the induction of RAR-ß expression and subsequent molecular signaling pathways to regulate the homeostasis of biochemical changes. This study demonstrated the capability of ATRA to prevent the establishment of metastasis by the melanoma cell line into the lung and liver of experimental mice.

The Effect of Indian Fig Fruit Extract on Human Papilloma Virus containing Cervical Cancer Cells (HeLa) by Decreasing the HPV18 L1 Gene Load.

BACKGROUND: Global trend is moving towards the use of natural phytochemicals to fight against pathogens. Human cervical cancer is directly associated with onco-potent type of Human Papilloma Virus (HPV). There is no known medicine for clearance of HPV type whose persistence is the cause of occurrence and re-occurrence of cervical cancer. The different species of fig fruit and their latex are reported to have HPV associated genital warts clearance capability. METHODS: In the current investigation, the effect of the methanol extract of Ficus benghalensis L. fruits on HPV type18 viral load in HeLa cell line was tested by doing PCR using HPV L1 primers (MY09/My011) and the cytotoxicity was also analysed by MTT assay. The induction of apoptotic activity in terms of DNA fragmentation and hyper-chromic effects of DNA was analysed. RESULTS: The PCR results showed a reduction in the HPV18 DNA and also the treatment exhibited a promising cytotoxicity with IC50 value at 211.86 µg/ml. The DNA samples from treated HeLa cells showed DNA shearing and laddering as a mark of apoptotic DNA fragmentation (Fig. 2) and the UV absorbance value at 260 nm was found to be significantly (p <0.01) higher in the DNA sample treated with fruit extract compared to the untreated DNA sample. CONCLUSION: The Ficus benghalensis L. fruit extract reduced the HPV viral load in HPV18 containing HeLa cells and showed an effective cytotoxicity on HeLa cell line. It also could induce the apoptotic activity in HeLa cell line and this study results suggest that the Ficus benghalensis L. fruits can be used to fight against cervical carcinoma, acting on HPV load.

Synergistic effect of co-treatment with all-trans retinoic acid and 9-cis retinoic acid on human lung cancer cell line at molecular level.

The major challenge in treating cancers with ATRA is the limited availability inside the cell and resistance developed in prolonged treatment. We made an attempt for co-treatment of human NSCLC cell lines (A549) with ATRA and its isomeric precursor (9cisRA). In this study, the growth inhibitory effect of ATRA, 9cisRA and combination of both were tested in A549 cells by MTT and Trypan blue assays. As the effects of retinoid are mediated through their receptors, their gene expression levels were analyzed by RT-PCR. The target gene receptor, RAR-ß protein expression, was analyzed by immunocytochemistry. The cancer cell (A549) growth inhibitory effect was significantly (p ≤ 0.001) enhanced in combination treatment when compared with the result of individual treatments. The mRNA expression levels of both RAR-ß and RXR-ß were found to be increased in co-treatment (band density of 0.75 and 0.806, respectively) when compared with 9cisRA treatment (0.25 and 0.112) and ATRA treatment (0.01 and 0.081). A concomitant enhancement in the target RAR-ß protein expression was observed in co-treated cells when compared with individual treatments. We thus conclude that the co-treatment had increased the availability of ATRA, by isomerization of the 9cisRA which then resulted in an increased expression of both RAR-ß and RXR-ß receptors and the target protein RAR-ß which in turn inhibited lung cancer cell growth. Our study results have explored the mechanism of synergistic effect of co-treatment with ATRA and 9cisRA and further preclinical studies are necessary to validate the application of co-treatment of retinoid in clinical use.

Liposome encapsulated all trans retinoic acid (ATRA) has enhanced immunomodulatory and inflammation reducing activities in mice model.

The all trans retinoic acid (ATRA) is found to have a promising regulatory effect on immune system and inflammatory responses in experimental research. The purpose of this study was to investigate whether this therapeutic efficiency of ATRA could be enhanced by encapsulating into a liposome formulation composed of Distearoyl-L-phosphatidylcholine (DSPC) and cholesterol utilizing a well-established mice model. The humoral antibody titer (HA), delayed-type hypersensitivity (DTH), bone marrow cellularity, hematology, and levels of α- esterase-positive cells, were taken as parameters to assess the level of immunomodulation in the sheep red blood cells (SRBC) immunized and challenged BALB/c mice. The anti-inflammatory effect of encapsulated ATRA was evaluated by the size changes in the induced inflammation edema in the mice paw as well as its histopathology. The results showed a significant immunostimulatory effect for both the free and encapsulated ATRA as indicated by the increase in the levels of total leukocyte, bone marrow and α-esterase positive cells and decreased Hb level respectively. We have also observed an enhanced specific antibody hemagglutinin titre value and the DTH response developed in response to SRBC challenge in these treatments. Both the immunostimulatory as well as inflammation reducing property were significantly higher in encapsulated ATRA treated group of mice over that of in free ATRA treated group of mice. Based on these results, we conclude that the encapsulated ATRA has a higher potency over free ATRA in its immunomodulatory activity and also has a significant impact on reducing inflammation in BALB/c mice model.

Anti-metastatic study of liposome-encapsulated all trans retinoic acid (ATRA) in B16F10 melanoma cells-implanted C57BL/6 mice.

B16F10 cells-induced C57BL/6 mice were divided into several groups and the free all trans retinoic acid (ATRA) and liposome-encapsulated ATRA were given for 21 days. The encapsulated ATRA treatment lowered the oxidative stress and lipid profile near to the normal level in the drug-treated mice. Encapsulated ATRA treatment showed substantial decrease in serum cytokines and increase in lifespan when compared with free ATRA treatment. These results imply that the liposome-encapsulated ATRA may help to achieve a higher level of ATRA in comparison with free ATRA treatment and helps to enhance anticancer drug delivery in liposome-encapsulated ATRA treatment.

Antioxidant potential of all-trans retinoic acid (ATRA) and enhanced activity of liposome encapsulated ATRA against inflammation and tumor-directed angiogenesis.

The purpose of this study was to investigate whether all-trans retinoic acid (ATRA) has antioxidant property. The study was also focused on its inhibitory effect on the acute and chronic inflammation and tumor-associated capillary formation in terms of angiogenesis in C57BL/6 mice after incorporated in liposome composed of distearoylphosphatidylcholine (DSPC/cholesterol). ATRA possesses a number of important biologic activities including oncostatic, antioxidant and immunostimulatory actions. Our study was designed to evaluate the antioxidant activity of free ATRA by nitric oxide scavenging, superoxide radical scavenging, hydroxyl radical scavenging and lipid peroxide scavenging assays. The ATRA showed significant scavenging activities in all these antioxidant assays comparable to the standard antioxidant. We have also evaluated the activity of encapsulated ATRA against anti-inflammatory activity in C57BL/6 mice. The paw oedema inhibition was found in carrageenan model as 55.56% and 66.67% for free ATRA and encapsulated ATRA treatment respectively and for formaldehyde model it was found to be 60.87% and 69.57% respectively compared with saline treated control mice. Encapsulated ATRA inhibited the tumor-associated capillary formation in mice induced by highly metastatic B16F10 melanoma cells significantly than the free ATRA did. In this study the inhibition of tumor-directed capillary formation was found to be 56.25% and 62.50% for free ATRA and encapsulated ATRA treatment respectively. In conclusion, ATRA showed a significant antioxidant property in vitro. Free ATRA has anti-inflammatory activity as proved by us in animal model of acute and chronic inflammation and antiangiogenesis activity. Furthermore, its activity was boosted by encapsulation in liposome.

The chemotherapeutic effect of essential oil of Plectranthus amboinicus (Lour) on lung metastasis developed by B16F-10 cell line in C57BL/6 mice.

Current investigation is to evaluate the anticancer activity of the essential oil of Plectranthus amboinicus (Lour) on B16F-10 melanoma cell line injected C57BL/6 mice, and it was simultaneously treated with the essential oil of P. amboinicus (Lour) (50 µg/dose) via i.p. for 21 days. The present investigation exhibited the potent chemotherapeutic/chemopreventive effect of the essential oil of P. amboinicus (Lour) over lung metastasis that developed. To our knowledge, this is the first report in evaluating the effect of essential oil of P. amboinicus (Lour) using lung cancer model.

Targeting tumor metastasis by regulating Nm23 gene expression.

The Nm23 gene is a metastatic suppressor identified in a melanoma cell line and expressed in different tumors where their levels of expression are associated with reduced or increased metastatic potential. Nm23 is one of the over 20 metastasis suppressor genes (MSGs) confirmed in vivo. It is highly conserved from yeast to human, implying a critical developmental function. Tumors with alteration of the p53 gene and reduced expression of the Nm23 gene are more prone to metastasis. Nm23-H1 has 3'-5' exonuclease activity. This review focuses on the role of Nm23 in cancer progression and also a potential novel target for cancer therapy.

Inhibition of metastatic lung cancer in C57BL/6 mice by liposome encapsulated all trans retinoic acid (ATRA).

The purpose of this study was to investigate whether all trans retinoic acid (ATRA) incorporated in liposome composed of distearoylphosphatidylcholine (DSPC/cholesterol) could inhibit the metastatic lung cancer in mice more efficiently than free ATRA. Metastatic lung cancer model was developed by intravenous injection of B16F10 cells and it is also referred as melanoma model. In this present study, C57BL/6 mice were divided into several groups as per experimental design and the free ATRA and liposome encapsulated ATRA were given for 21 days at a dose of 0.60 mg/kg body weight/day after cell line implantation. After 21 days, mice were sacrificed at different time interval for ATRA level analysis in serum and lung tissue by HPLC method and the remaining mice were kept for anticancer study. The ATRA level increased significantly in serum and lung tissue in liposome encapsulated ATRA treated mice. In cancer bearing mice, tumor nodule formation decreased and life span increased after receiving liposome encapsulated ATRA treatment than free ATRA treated mice. This result implies that the liposome encapsulated ATRA has maintained more ATRA concentration in lung tissue and showed more inhibition on the lung tumor nodule formation. The results indicate a possible use of liposome encapsulated ATRA in prevention of lung metastasis.

Effect of all-trans retinoic acid (ATRA) on syndecan-1 expression and its chemoprotective effect in benzo(α)pyrene-induced lung cancer mice model.

All-trans retinoic acid (ATRA), an active metabolite of retinal, has been shown to exert anti-cancer activities in a number of cancer cells and tissues. Syndecan-1 is a proteoglycan, mediate cell-cell adhesion and prevent invasion in epithelial cells. The aim of the present study was to examine the level of syndecan-1 expression and the chemopreventive effect of ATRA during lung cancer development in BALB/c mice. Syndecan-1 expression was examined by immunohistochemistry using mouse monoclonal anti-human syndecan-1 antibody. In this study, benzo(α)pyrene [B(α)P] was used to induce lung cancer. The results indicated that ATRA has anti-cancer effect against B(α)P-induced lung tumor development as induced by number of tumor nodules and histopathologic report. The loss of syndecan-1 expression in the epithelial cell membrane is associated with tumor cell growth and invasiveness. Our study for syndecan-1 indicated a chemoprotective effect of ATRA against changes in lung epithelial cell membrane syndecan-1 expression in B(α)P-induced lung cancer model. Therefore ATRA could serve as effective chemotherapeutic agent against cancer invasion/metastasis, at least in the lungs.

Essential oil of Tridax procumbens L induces apoptosis and suppresses angiogenesis and lung metastasis of the B16F-10 cell line in C57BL/6 mice.

BACKGROUND: To determine the effect of essential oil obtained from a traditionally used medicinal plant Tridax procumbens L, on lung metastasis developed by B16F-10 melanoma cells in C57BL/6 mice. MATERIALS AND METHODS: Parameters studied were toxicity, lung tumor nodule count, histopathological features, tumor directed capillary vessel formation, apoptosis and expression levels of P53 and caspase-3 proteins. RESULTS: In vitro the MTT assay showed cytotoxicity was found to be high as 70.2% of cancer cell death within 24 hrs for 50 µg. In vivo oil treatment significantly inhibited tumor nodule formation by 71.7% when compared with untreated mice. Formation of tumor directed new blood vessels was also found to be inhibited to about 39.5%. TUNEL assays also demonstrated a significant increase in the number of apoptotic positive cells after the treatment. P53 and caspase-3 expression was also found to be greater in the essential oil treated group than the normal and cancer group. CONCLUSIONS: The present investigation showed significant effects of the essential oil of Tridax procumbens L in preventing lung metastasis by B16F-10 cell line in C57BL/6 mice. Its specific preventive effect on tumor directed angiogenesis and inducing effect on apoptosis warrant further studies at the molecular level to validate the significance of Tridax procumbens L for anticancer therapy.

Lysyl oxidase: a potential target for cancer therapy.

Lysyl oxidases (LysOX; EC 1.4.3.13, protein-lysine 6-oxidases) are extracellular copper enzymes that catalyze the cross-linking of collagens or elastin in the extracellular matrix (ECM), thereby regulating the tensile strength of tissues. Recent implication of LysOX in cancer, wound healing, cell motility, chemotaxis, and differentiation reflects its remarkable functional diversity and also in the central nervous system pathologies. However, recent reports also demonstrated novel roles for LysOX, including the ability to regulate gene transcription, motility/migration, and cell adhesion. These diverse functions have led researchers to hypothesize that LysOX may have multiple roles affecting both extra- and intracellular cell function(s). Both down and up-regulation of LysOX in tumor tissues and cancer cell lines have been described, suggesting a dual role for LysOX as a tumor suppressor, as well as a metastasis promoter gene. In this review we explain in detail the role of lysyl oxidase in tumor progression and metastasis.

Co-overexpression of p53 and bcl-2 proteins in HPV-induced squamous cell carcinoma of the uterine cervix.

OBJECTIVE: The aim of this study was to analyze aberrant expression of both apoptotic protein p53 and antiapoptotic protein bcl-2 in squamous cell carcinoma (SCC) of the uterine cervix with HPV infection and its significance as a marker for progression of cervical lesions. METHODS: One hundred and five cervical lesions and 20 normal (age matched) cervical epithelium from patients with complaints other than cervical lesions were investigated immunocytochemically for aberrant expression of p53 and bcl-2 using the streptavidin-biotin-peroxidase method with respective monoclonal antibodies. HPV status was also anlayzed using type-specific primers for HPV 16/18 and HPV 6/11 by polymerase chain reaction (PCR). The statistical correlation analysis was carried out using Spearman's correlation test and univariate analysis by the SPSS system. RESULTS: An abnormal nuclear expression of tumor-suppressor protein p53 and cytoplasmic expression of bcl-2 were observed using immunocytochemistry in biopsies of cervical lesions but not in normal subjects. The intensity of immunoreactivity for both p53 and bcl-2 proteins varied between different histopathological grades of cervical lesions and the correlation analysis showed a highly significant positive correlation for their expression level with different stages from mild dysplasia to invasive cancer with r = 0.88842; P = 0.00001 and r = 0.86929; P = 0.00001, respectively. A highly significant positive correlation was also observed between the expression of both p53 and bcl-2 proteins and HPV infection. The current study indicates a very good significant direct correlation (r = 0.83925; P = 0.00001) between p53 expression and bcl-2 expression in the study population, suggesting the co-overexpression of these proteins in HPV-associated cervical cancer. CONCLUSIONS: From the observations it is suggested that the immunodetection of both p53 and bcl-2 proteins in squamous cell carcinoma of the uterine cervix can be used as an independent diagnostic marker for cervical cancer associated with HPV infection. The highly significant association of these proteins with HPV infection suggests that the high-risk HPV infection may be responsible for the co-overexpression of p53 and bcl-2 in cervical cancer even though both of them are antagonistic in their function. This study thus helps to understand the molecular mechanism underlying cervical carcinogenesis and which in turn may improve the therapeutic approach.