Amelioration of Combination of Paclitaxel and Di Allyl Sulfide on the Alterations of Bcl2, P53 and Apoptosis Changes Against 7,12 Di Methyl Benz (A) Anthracene Induced Skin Cancer in Experimental Animals

Abstract

The purpose of this study was to investigate the Bcl2, P⁵³ and apoptosis changes against skin cancer in experimental animals. Skin cancer is the most common form of human cancer. It is estimated that over 1 million new cases occur annually. The annual rates of all forms of skin cancer are increasing each year, representing a growing public concern. It has also been estimated that nearly half of all Americans who live to age 65 are likely to develop skin cancer at least once. Skin cancer was induced in rats by Di Methyl Benz (a) Anthracene at the dosage of DMBA (5 µg) per animal, three times a week for 28 weeks after conformation of skin cancer treated with Paclitaxel and Di allyl sulfide for 30 days. The levels of Bcl2 gene expression were significantly decreased and P⁵³gene expression were markedly increased in Paclitaxel and Di allyl sulfide treated animals when compared with cancer bearing animals. The treatment with combination of Paclitaxel and Di allyl sulfide effectively reduced Bcl2 protein expression and also increased P⁵³gene expression. Moreover, the levels of Bcl2 and P⁵³ a good indicators of restoring the skin architecture, were also reversed in skin damage subjects after treatment with the herbal compounds preparation. So, from the obtained results it is concluded that a combination of Paclitaxel and Di allyl sulfide is capable of restoring the skin architecture and can also increase the apoptosis activities in skin cancer rats.

Introduction

In India, Skin cancers constitute about 1–2% of all diagnosed cancers. Basal cell carcinoma is the commonest form of skin cancer worldwide, but various studies from India have consistently reported SCC as the most prevalent skin malignancy. Although complete data of incidence is not available, various cancer registries in India reported cumulative incidence of skin cancer varying from 0.5 to 2 per 100,000 population [1]. Although, the incidence of skin cancers in India is lower as compared to the Western world, because of a large population, absolute number of cases is estimated to be significant. In this research our goal was to establish whether the Bcl2, p53 and apoptosis can be used as a new gene for the assessment of carcinogenic risk of skin cancer. Polycyclic aromatic hydrocarbons (PAH) are an important class of direct and indirect chemical carcinogens. PAH bind to DNA by two major mechanisms of activation: one-electron oxidation to form radical cations [2] and monooxygenation to form diol epoxides [3]. DMBA, a potent PAH recognized as an initiator of both skin and liver cancer [4]. The covalent binding of DMBA metabolites to DNA has been implicated as a critical step in the initiation phase of cancers. In skin, 99% of the DMBA-DNA adducts are depurinating adducts formed by one-electron oxidation, in which the 12- methyl group of DMBA reacts with the N-7 of adenine or guanine in a 4:1 ratio, respectively. Only a fraction of the 1% of stable adducts corresponds to diol epoxide products. Paclitaxel (Taxol), a naturally occurring antineoplastic agent has shown great promise in the therapeutic management of certain human solid tumors particularly in metastatic breast cancer and malignancy involves skin, lung and refractory ovaries. It is the original member of the taxane group of anticancer drugs derived from the bark and needles of the pacific yew tree “Taxus brevifolia”. Paclitaxel’s antitumor activity was discovered in 1960’s during a large scale 35,000 plants-screening program sponsored by the National Cancer Institute (NCI), USA. The chief constituent of garlic is the sulfur compound allicin, produced by crushing or chewing fresh garlic, which in turn produces other sulfur compounds: ajoene, mono-, di-, and tri-allyl sulfides, and vinyldithiins [5]. Di allyl sulfide one of the active form of Allium vegetable- derived organosulfur compound and directly involved cell cycle regulation. Apoptosis that occurs as part of normal development is often referred to as programmed cell death (PCD). Programmed cell death is regulated by two major families of proteins including proapoptotic members and anti-apoptotic members of the Bcl-2 family [6,7], and caspases [8, 9]. Proapoptotic Bcl-2 family members can be divided into two groups. Members of the first group all have at least two of the four Bcl-2 homology domains (BH1 to 4) found in Bcl-2 while the second group only contain the BH3 domain.

On the basis of this theoretical background the following investigation will include: Bcl2 and P53 apoptosis, to ascertain the role of Paclitaxel and Di Allyl Sulfide combination in decreasing the macromolecular damages and improving the apoptosis in experimentally induced skin cancer rats.

Materials and Methods

Chemicals

7,12 Dimethyl benz (a) anthracene and Di allyl sulfide were purchased from Sigma chemical company, USA. All the other chemicals used were of analytical grade.

Animal Care and Housing

Male Wistar rats, 6–8 weeks of age and weighing 150–200 g, were used. The animals were procured from Central Animal House Block, Meenakshi Medical College and Research institute, Kanchipuran, Tamil Nadu, India and maintained in a controlled environmental condition of temperature and humidity on alternatively 12 h light/dark cycles. All animals were fed standard pellet diet (Gold Mohor rat feed, Ms. Hindustan Lever Ltd., Mumbai) and water ad libitum. This research work on Wistar male rats was sanctioned and approved by the Institutional Animal Ethical Committee (REG NO. 765/03/ca/CPCSEA).

Experimental Design

The animals were divided into six groups of 6 animals each. Group I animals served as control, Group II, III, IV, V as animals treated with DMBA (5 µg) per animal in acetone (100 µL), three times a week for 28 weeks to induce skin cancer. After tumor induction Group III animals were treated with Paclitaxel (33 mg/kg b.wt) once in a week for 4 weeks. Group IV animals were treated with garlic extract of Di allyl sulfide (250 µg/kg b.wt) for 30 days. Group V animals were treated with both Paclitaxel and Di allyl sulfide (as in group III and group IV). This was Group VI Control animals treated with paclitaxel and Di allyl sulfide for 28 weeks plus 30 days. Di allyl sulfide dosing concentration 250 µg/kg b.wt was fixed by dose dependent study. The animals were treated with six different dose concentration started from 50 µg, 100 µg, 150 µg, 200 µg, 250 µg and 300 µg/kg b.wt. A dosing concentration of 250 µg/kg b.wt of Di allyl sulfide is highly responsible to treat the cancer induced wistar rats and its conformed with biochemical markers like antioxidants and lipid metabolizing enzyme activity.

After the experimental period of 32 weeks, the animals were sacrificed by cervical decapitation.

Immnoblotting Analysis for Bcl-2 and p53

Skin cancer cells (5 × 10⁵) were seeded into 6- well plates, grown for 24 h, and incubated in the presence or absence of drug. Cells were collected, washed twice with ice cold PBS and lysed with 50 mM Tris HCl (pH 8.0), containing 150 mM NaCl, 1% Triton X-100, 100 µg/ml phenylmethylsulfonyl fluoride and 1 µg/ml aprotinin. This was followed by incubation for 30 min in ice and then centrifuged at 10,000 × g for 20 min at 4 °C. Supernatant was collected and used for western blotting. The protein concentration was measured by Lowry’s method [10].

Bcl-2 (50 μg of protein) and p53 (40 μg of proteinprotein were fractionated by SDS-PAGE using 12% gel and transferred to nitrocellulose membrane. The stacking gel was 4% and the resolving gel was 12%. After electrophoresis, the gel was placed over an appropriately cut nitrocellulose (NC) membrane. The gel and the PVDF membrane were packed by three cut-pieces of Whatmann filters (No. 3). This set-up was covered on both sides with the absorbers (provided with the system) and clipped. The whole set-up was immersed in a tank containing blotting buffer. A current of 100 V was passed through for 60 min. The membrane was then removed from the system and immersed in methanol for a minute. Empty sites in the membrane were blocked by treating the membrane with blocking solution for 1 h at 37 °C. After washing, the membrane was incubated with primary antibody, for 1 h at 37 °C. Then the wells were washed, charged with 100 μl of secondary antibody and incubated at 37 °C for 1 h. Again after washing, the activity of bound ALP was monitored by the addition of 100 μl of Biotin/NBT reagent. In case of HRP bound secondary antibody, HRP activity was monitored by treating the membrane with 3, 3-diaminobenzidine tetrahydrochloride (DAB) reagent and 0.01% H₂O₂ in 0.05 M Tris—HCl buffer. After the development of colors, the membrane was washed in water and analyzed for changes.

Analysis of Gene Expression by Semi-quantitative RT-PCR

Isolation of RNA

Total RNA extraction from skin was performed using Trizol reagent, which is based on the acidic phenol chloroform method [11]. About 50 mg of minced skin tissue was homogenized in 1.0 ml of ice cold Trizol reagent (Sigma). The homogenate was cleared off by centrifugation at 12,000 rpm for 5 min. To the supernatant 0.2 ml of ice cold chloroform was added and shaken vigorously for 2 × 10 s. The contents were centrifuged at 12,000 rpm for 15 min, decanted and the supernatant carefully transferred into a fresh tube. To this two volumes of ice cold isopropanol was added and mixed gently by repeated inversion of the tubes. RNA precipitation was enhanced by storing at − 20 °C for ~ 3 h. After incubation, RNA was pelleted out by centrifugation at 12,000 rpm. The RNA pellet was washed once with 70% ice cold ethanol and allowed to air dry to remove ethanol. The pellet was resuspended in 50 μl of RNase free-H₂O and stored at − 80 °C. All centrifugation steps were carried out at 4 °C unless otherwise stated. (Note: All the containers/materials were rinsed with 0.1% DEPC-water and autoclaved before use).

Determination of RNA Purity and Concentration

The final preparation of total RNA is essentially free of DNA and proteins and has a 260/280 ratio of 1.6–1.8.

$$\text{RNA concentration}\left(\mathrm{\mu }\text{g}/\text{ml}\right)={\text{OD}}_{260}\times 40\times \text{dilution factor}/1\left({\text{OD}}_{260}\right)$$

where 40 = standard quantity of RNA/1 (OD₂₆₀), Dilution factor = 100 (10 µl made up to 1.0 ml).

Reverse Transcription–Polymerase Chain Reaction (RT–PCR) cDNA Synthesis

Total RNA (1 μg) was subjected to a total volume of 20 μl RT–PCR containing 5 μl of 10X RT-buffer, 10 mm dNTPs, cDNA synthesis mix (50 μM of Oligo (dT)₂₀, 25 mm MgCl₂, 0.1 M DTT, 1 μl RNase out (40 U/μl), 1 μl SuperScript-RT). cDNA synthesis is achieved in three steps; initially the reaction was carried out with the RNA, dNTPs and buffer alone at 65 °C for 5 min followed by the cDNA synthesis mix were added and kept for 50 min at 50 °C. The reaction was arrested by heating at 85 °C for 10 s followed by degradation of the remaining RNA by incubation with RNase H for 20 min at 37 °C. Then, cDNA was subjected to PCR for the analysis of various gene expressions.

Preliminary experiments were conducted with each gene to ensure that the number of cycles represented a linear portion for the PCR OD curve for the skin samples. After amplification, the RT–PCR products were electrophoresed on 2% agarose gels, and stained with ethidium bromide. Images were captured and subjected to densitometric analysis. RPL19 and β-actin were used as internal controls, while all RT–PCR signals were normalized to either RPL19 or β-actin signal of the corresponding product to eliminate the measurement error from uneven sample loading, and to provide a semi-quantitative measure of the relative changes in gene expression. The values were expressed as percentage ratio of target genes signal intensity relative to RPL19 or β-actin.

Analysis of Protein Expression by Immunoblotting

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) was performed for the electrophoretic separation of tissue proteins [12]. By mixing the solution of proteins with SDS, protein is denatured and gets a negative charge due to binding of SDS to the protein giving an approximately uniform mass: charge ratio. This enables the proteins to be resolved strictly based on the molecular mass. With the addition of SDS, proteins were briefly heated to 95 °C in the presence of a reducing agent (Dithiothreitol or β-mercaptoethanol) for further denaturation. The denatured proteins were subsequently applied to one end of a layer of polyacrylamide gel submerged in a suitable buffer and an electric current was applied across the gel causing the negatively-charged proteins to migrate depending on their size. In SDS-PAGE, the protein separation was performed using a discontinuous buffer system. In early stage of electrophoresis, an ion gradient is formed in the stacking gel that causes all of the proteins to focus into a single sharp band. With the change of pH and the subsequent elimination of the ion gradient in the resolving gel causes the proteins to separate by the molecular size sieving.

A system with vertical oriented glass plates (10 × 10 cm; 1 mm spacer) was used. The gels were prepared by casting the resolving gel (10 or 12% acrylamide) between glass plates and covered with a layer of butanol. After polymerisation of the gel was completed, butanol was removed and the space between glass plates was dried with Whatman paper. Then the stacking gel was cast on top of the polymerised resolving gel and the sample loading comb was introduced immediately. After completion of the polymerisation the sample loading comb was removed. The glass plates with the gel between them were fixed inside the electrophoresis chamber and covered with electrophoresis buffer. Protein samples were mixed with Laemmli loading buffer 3:1 ratio, denatured by boiling the sample at 95 °C for 10 min followed by brief cooling on ice and centrifugation at 14,000 rpm. The clear supernatant was applied into the stacking gel pockets. Electrophoresis was performed for 2–3 h at a constant voltage of 75–100 V.

The protein-transferred membrane was blocked in PBS-Tween with 10% non-fat milk powder for 3 h at room temperature on a shaker. The blocked membrane was incubated with suitably diluted primary antibodies for 3 h at room temperature on a shaker. After incubation, the membrane was washed with TBS-Tween 3 × 10 min. The membrane was incubated for at least 45 min with suitably diluted secondary antibodies at room temperature on a shaker. The membrane was alternatively washed with 3 × 10 min each with TBS-Tween and TBS. Then the membrane was developed using the sensitive chemiluminescence method (Pierce). The signal was detected with X-ray film and analyzed using multi analyst (Bio-Rad).

Statistical Analysis

One-way Analysis Of Variance (ANOVA) and Tukey’s Multiple Comparison Test was done to evaluate the significance of difference of means of various treatment groups, using SPSS (Statistical Package for Social Science) statistical package (Version: 17). The data were also subjected to Student‘t’ test (Paired) wherever necessary, to evaluate the significance of difference of means of the control and experimental groups using SPSS software. The values are presented as mean ± S.D. and p value < 0.05 was considered significant [13].

Results

Apoptosis

The Fig. 1 (Western blot analysis) shows the expression of Bcl2 protein in control and experimental animals. In cancer bearing animals (G-II) there was found to be a significant expression of Bcl2 protein when compared with control animals (G-I). This was significantly (P < 0.05) decreased the Bcl2 levels in animals subjected to combination of paclitaxel and Di allyl sulfide (G-V) when compared with the cancer-induced group. However, there was no much of difference in animals treated with paclitaxel and Di allyl sulfide (G-VI) when compared with control animals. Similar results were noted in Western blot analysis in treating skin cacer cells with paclitaxel and Di allyl sulfide. The result thereby suggests combination treatment of paclitaxel and Di allyl sulfide are effective in decreasing tumor growth.

Fig. 1.

Effect of paclitaxel and paclitaxel—di allyl sulfide on protein and gene expression of Bcl₂ in control and experimental animals (Western blot analysis—Bcl₂). Lane 1: marker, Lane 2: control, Lane 3: DMBA treated, Lane 4: paclitaxel treated, Lane 5: Di allyl sulfide treated, Lane 6: paclitaxel and Di allyl sulfide treated cancer bearing animal, Lane 7: paclitaxel and Di allyl sulfide treated control animal

The Fig. 2 (Western blot analysis) shows the level of p53 gene expression by skin cancer cells on drug treatments when analyzed by Western blot analysis. The level of p53 protein was found to be up-regulated in skin cancer cells when treated with paclitaxel and Di allyl sulfide by P < 0.05, indicating arrest of cell cycle. The combination treatment of paclitaxel and Di allyl sulfide was proved to be significant (P < 0.05) than individual drug treatments in elevating the p53 protein expression levels.

Fig. 2.

Effect of paclitaxel and paclitaxel—Di allyl sulfide on protein and gene expression of P⁵³ in control and experimental animals (Western blot analysis—P⁵³). Lane 1: marker, Lane 2: control, Lane 3: DMBA treated, Lane 4: paclitaxel treated, Lane 5: Di allyl sulfide treated, Lane 6: paclitaxel and Di allyl sulfide treated cancer bearing animal, Lane 7: paclitaxel and Di allyl sulfide treated control animal

Rt-pcr

Reverse transcription polymerase chain reaction (RT-PCR) amplifies a defined piece of RNA into its DNA complement, followed by amplification of the resulting DNA using polymerase reaction. Thereby RT-PCR helps us to determine the level of gene expressed in the cells of interest.

Bcl-2

The major anti-apoptotic members of the Bcl-2 family the Bcl-2 protein, has a crucial role in intracellular apoptotic signal transduction. Figure 3 shows the level of Bcl-2 gene expression in skin cancer cells on drug treatments. Increased level of Bcl-2 gene was expressed in skin cancer animals cells when compared with the control group, indicating tumor growth. However, combination of paclitaxel and Di allyl sulfide treatments to skin cancer cells significantly decreased (P < 0.05) the level of Bcl-2.

Fig. 3.

Effect of paclitaxel and paclitaxel—Di allyl sulfide on protein and gene expression of Bcl₂ in control and experimental animals (Bcl2 RT-PCR). Lane 1: marker (100 bp), Lane 2: control, Lane 3: DMBA treated, Lane 4: paclitaxel treated, Lane 5: Di allyl sulfide treated, Lane 6: paclitaxel and Di allyl sulfide treated cancer bearing animal, Lane 7: paclitaxel and Di allyl sulfide treated control animal

p53

The p53 protein acts as a checkpoint in the cell cycle, either preventing or initiating programmed cell death. The loss of this protein due to mutation is a primary event in the formation of many types of cancer (skin, breast, colon, lung, and leukaemia).

Figure 4 shows the level of p53 gene expression in skin cancer cells on drug treatments. Decreased level of p53 gene was expressed in skin cancer bearing animal when compared with the control group. However, combination of paclitaxel and Di allyl sulfide treatments to skin cancer cells significantly increased (P < 0.05) the level of p53.

Fig. 4.

Effect of paclitaxel and paclitaxel—Di allyl sulfide on protein and gene expression of P⁵³ in control and experimental animals (P⁵³ RT-PCR). Lane 1: marker (100 bp), Lane 2: control, Lane 3: DMBA treated, Lane 4: paclitaxel treated, Lane 5: Di allyl sulfide treated, Lane 6: paclitaxel and Di allyl sulfide treated cancer bearing animal, Lane 7: paclitaxel and Di allyl sulfide treated control animal

Results for the effect of Paclitaxel and Paclitaxel+Di Allyl Sulfide on the p⁵³ and Bcl2 protein concentration in control and experimental animals are depicted in the Fig. 5

Fig. 5.

Each Values expressed as Mean + SD for Six rats in each Group a- as compared with Group-II cancer bearing rats. Statistical Significance—*P < 0.001

In the present study, indicates that the concentration of apoptotic protein p53 and anti apoptotic protein Bcl2 in skin lesions in different groups. P53 protein concentration were significantly (P < 0.001) increased in Group-II cancer bearing rats when compared with Group-III, Group-IV and Group-V cancer treated rats. On the other hand, anti-apoptotic proteins of Bcl2 levels were significantly (P < 0.001) decreased in cancer bearing rats when compared with skin cancer treated groups of III,IV and Group-V. But there was no significantly changes in Group-VI rats compared with group-I control rats.

Discussion

Apoptosis

Apoptosis is a complex process that removes the injured cells from the body and occurs in a wide variety of organisms. Cell death has always been an integral aspect of the study of pathology, but only over the last 30 years the interest in apoptosis gained appreciation in this field [14].

Apoptosis or programmed cell death is a genetically regulated cellular, physiological and biochemical suicidal mechanism that plays a crucial role in the development of homeostasis. It is currently hypothesized that tumor development is a result of an alteration in cell death[15]. Cancer development is known to be associated with increased cell proliferation and decreased apoptosis. Induction of apoptosis serves as a mean to suppress or reverse cancer development [15, 16]. Most of the chemotherapeutic agents either prevents cell proliferation or involve in the genetically regulated cellular process of programmed cell death. The success of this approach relies on the ability to manipulate tumor cells to commit to the death program[17].

Apoptosis event includes DNA fragmentation, chromatic condensation, membrane blebbing, cell shrinkage and disassembly into membrane-enclosed vesicles. These changes occur in a predictable, reproducible sequence and can be completed within a minute [18].

Bcl2 and Apoptosis

The expanding families of Bcl oncogenes are among the main regulators of apoptosis. Some of its members, Bcl2, Bcl1 and Bcl-XL, function as blockers of cell death, while others, bax, bak and Bcl-XS, function as promoters of apoptosis [19]. The name Bcl2 (B cell lymphoma/leukemia gene 2) signifies the close association of this gene to these malignancies in which enhanced expression was initially believed to arise solely as a result of these translocations, resulting in the juxtaposition of the Bcl2 gene to a potent enhancer element sequence of the Ig H gene. Several genes have been identified and designated as the Bcl2 family based on their sequence homology to Bcl2. These genes include both positive and negative regulators of apoptosis [20]. The proto-oncogene Bcl2 protects cells against apoptosis. Morphological and biochemical studies demonstrate that Bcl2 has two major intracellular localizations: (i) mitochondria and (ii) the endoplasmic reticulum (ER). Mitochondria have received major attention as organelles involved in apoptosis. However, Bcl2 not only prevents the mitochondrial permeability switch and the subsequent release of cytochrome C, but also inhibits apoptosis in response to cytochrome C. This suggests that Bcl2 inhibits apoptotic mechanisms downstream of cytochrome C, possibly at the level of the ER. Consistent with this hypothesis, a study demonstrated that ER-targeted Bcl2 was able to inhibit apoptosis induced by myc in a rat fibroblast cell line [21].

In vitro studies have highlighted the role of Bcl2 proteins as important regulators of the apoptotic pathway in several cell types. Bcl2, the prototype of this family was discovered by studies of translocations, which are frequent in Non-Hodgkin lymphomas and follicular lymphomas [20].

Paclitaxel is a drug highly effective in the treatment of several neoplasms, including ovarian, breast, lung and skin carcinomas. Paclitaxel treatment strongly inhibited Bcl2 protein expression in the treated mice. Although the precise means by which cell death occurs are not clear, DNA fragmentation patterns characteristic of apoptosis have been documented after paclitaxel treatment of tumor cells. The apoptotic effects of paclitaxel have been associated with phosphorylation of Bcl2, an antiapoptotic protein. However, Bcl2 protein down-regulation can also result in induction of apoptosis [21]. The skin cancer bearing animals treated with combination of paclitaxel and DAS also caused down regulation of Bcl2 in the present study. Thus from the present study result, it was postulated that induction of apoptosis was involved in the antitumor activity of DAS.

p53 and Apoptosis

p53 (also known as protein 53 or tumor protein 53), is a transcription factor encoded by the TP53 gene. p53 is important in multicellular organisms, where it regulates the cell cycle and thus functions as a tumor suppressor that is involved in preventing cancer. The p53 gene has been mapped to chromosome 17. In the cell, p53 protein binds DNA, which in turn stimulates another gene to produce a protein called p21 that interacts with a cell division-stimulating protein (cdk2). When p21 is complexed with cdk2 the cell cannot pass through to the next stage of cell division. Mutant p53 can no longer bind DNA in an effective way, and as a consequence the p21 protein is not made available to act as the ‘stop signal’ for cell division. Thus cells divide uncontrollably, and form tumors. Exposure to cellular stress can trigger the p53 tumor suppressor, a sequence-specific transcription factor, to induce cell growth arrest or apoptosis. The choice between these cellular responses is influenced by many factors, including the type of cell and stress, and the action of p53 co-activators.

In our present study, the level of p53 expression was found to be significantly decreased in skin cancer cells. This might be due to the decrease in cell cycle arrest leading to proliferation of skin cancer cells. While, Immunoblot analysis from skin cancer cells treated with paclitaxel and Di allyl sulfide individually and in combination demonstrated significant up regulation of p53 levels. The reason behind it could be possibly due to the influence of paclitaxel induced mitotic arrest and anti-tumor properties of Di allyl sulfide in increasing the cell cytotoxic effects on skin cancer cells. Furthermore, recent studies have revealed that paclitaxel-induced apoptosis was concentration- dependent and cell cycle dependent and low dose (0.01 µM) paclitaxel- induced apoptotic processes were mediated in a p53 signaling pathway [22]. Paclitaxel also caused p53 upregulation in the apoptosis-resistant SCC-VII tumors [23]. In the present study paclitaxel treatment at 0.5 µM up regulated p53 expression, but its effect on mediating apoptosis was not clear. Studies with Di allyl sulfide has reported that consistent with the occurrence of DNA fragmentation, nuclear p53 protein increases after Di allyl sulfide treatment, suggesting the p53-associated signaling pathway is critically involved in Di allyl sulfide -mediated apoptotic cell death [24, 25]. Thus Paclitaxel and Di allyl sulfide combination treatments are highly efficient in up regulating the p53 pathway and ultimately decreasing the survival of skin cancer cells.

Conclusion

From the experimental studies we suggest that combination of Paclitaxel and Di allyl sulfide is having a anticancer and apoptosis effect in cancer induced animals. Both Paclitaxel and Di allyl sulfide acts on Cell cycle. The results from our study suggest that Paclitaxel and Di allyl sulfide can be explored for its potential as a targeted therapy for skin cancer.