Selective apoptotic cell death effects of oral cancer cells treated with destruxin B.

BACKGROUND: Recent studies have revealed that destruxins (Dtx) have potent cytotoxic activities on individual cancer cells, however, data on oral cancer cells especial human are absent. METHODS: Destruxin B (DB) was isolated and used to evaluate the selective cytotoxicity with human oral cancer cell lines, GNM (Neck metastasis of gingival carcinoma) and TSCCa (Tongue squamous cell carcinoma) cells, and normal gingival fibroblasts (GF) were also included as controls. Cells were tested with different concentrations of DB for 24, 48, and 72 h by MTT assay. Moreover, the mechanism of cytotoxicity was investigated using caspase-3 Immunofluorescence, annexin V/PI staining, and the expression of caspase-3, Bax, and Bcl-2 by western blotting after treated with different concentrations of DB for 72 h as parameters for apoptosis analyses. RESULTS: The results show that DB exhibited significant (p < 0.01) and selective time- and dose-dependent inhibitory effects on GNM and TSCCa cells viability but not on GF cells. The data suggested that DB is capable to induce tumor specific growth inhibition in oral GNM and TSCCa cancer cells via Bax/Bcl-2-mediated intrinsic mitochondrial apoptotic pathway in time- and dose-dependent manners. CONCLUSIONS: This is the first report on the anti-proliferation effect of DB in oral cancer cells. The results reported here may offer further evidences to the development of DB as a potential complementary chemotherapeutic target for oral cancer complications.

Extracellular matrix-regulated neural differentiation of human multipotent marrow progenitor cells enhances functional recovery after spinal cord injury.

BACKGROUND CONTEXT: Recent advanced studies have demonstrated that cytokines and extracellular matrix (ECM) could trigger various types of neural differentiation. However, the efficacy of differentiation and in vivo transplantation has not yet thoroughly been investigated. PURPOSE: To highlight the current understanding of the effects of ECM on neural differentiation of human bone marrow-derived multipotent progenitor cells (MPCs), regarding state-of-art cure for the animal with acute spinal cord injury (SCI), and explore future treatments aimed at neural repair. STUDY DESIGN: A selective overview of the literature pertaining to the neural differentiation of the MSCs and experimental animals aimed at improved repair of SCI. METHODS: Extracellular matrix proteins, tenascin-cytotactin (TN-C), tenascin-restrictin (TN-R), and chondroitin sulfate (CS), with the cytokines, nerve growth factor (NGF)/brain-derived neurotrophic factor (BDNF)/retinoic acid (RA) (NBR), were incorporated to induce transdifferentiation of human MPCs. Cells were treated with NBR for 7 days, and then TN-C, TN-R, or CS was added for 2 days. The medium was changed every 2 days. Twenty-four animals were randomly assigned to four groups with six animals in each group: one experimental and three controls. Animals received two (bilateral) injections of vehicle, MPCs, NBR-induced MPCs, or NBR/TN-C-induced MPCs into the lesion sites after SCI. Functional assessment was measured using the Basso, Beattie, and Bresnahan locomotor rating score. Data were analyzed using analysis of variance followed by Student-Newman-Keuls (SNK) post hoc tests. RESULTS: Results showed that MPCs with the transdifferentiation of human MPCs to neurons were associated with increased messenger-RNA (mRNA) expression of neuronal markers including nestin, microtubule-associated protein (MAP) 2, glial fibrillary acidic protein, ßIII tubulin, and NGF. Greater amounts of neuronal morphology appeared in cultures incorporated with TN-C and TN-R than those with CS. The addition of TN-C enhanced mRNA expressions of MAP2, ßIII tubulin, and NGF, whereas TN-R did not significantly change. Conversely, CS exposure decreased MAP2, ßIII tubulin, and NGF expressions. The TN-C-treated MSCs significantly and functionally repaired SCI-induced rats at Day 42. Present results indicate that ECM components, such as tenascins and CS in addition to cytokines, may play functional roles in regulating neurogenesis by human MPCs. CONCLUSIONS: These findings suggest that the combined use of TN-C, NBR, and human MPCs offers a new feasible method for nerve repair.

In vitro antagonistic growth effects of Lactobacillus fermentum and Lactobacillus salivarius and their fermentative broth on periodontal pathogens

As lactobacilli possess an antagonistic growth property, these bacteria may be beneficial as bioprotective agents for infection control. However, whether the antagonistic growth effects are attributed to the lactobacilli themselves or their fermentative broth remains unclear. The antagonistic growth effects of Lactobacillus salivarius and Lactobacillus fermentum as well as their fermentative broth were thus tested using both disc agar diffusion test and broth dilution method, and their effects on periodontal pathogens, including Streptococcus mutans, Streptococcus sanguis, and Porphyromonas gingivalisin vitro at different concentrations and for different time periods were also compared. Both Lactobacillus salivarius and Lactobacillus fermentum and their concentrated fermentative broth were shown to inhibit significantly the growth of Streptococcus mutans, Streptococcus sanguis, and Porphyromonas gingivalis, althoughdifferent inhibitory effects were observed for different pathogens. The higher the counts of lactobacilli and the higher the folds of concentrated fermentative broth, the stronger the inhibitory effects are observed. The inhibitory effect is demonstrated to be dose-dependent. Moreover, for the lactobacilli themselves, Lactobacillus fermentum showed stronger inhibitory effects than Lactobacillus salivarius. However, the fermentative broth of Lactobacillus fermentum showed weaker inhibitory effects than that of Lactobacillus salivarius. These data suggested that lactobacilli and their fermentative broth exhibit antagonistic growth activity, and consumption of probiotics or their broth containing lactobacilli may benefit oral health.

Anticancer effects of eleven triterpenoids derived from Antrodia camphorata.

Eleven derivatives from Antrodia camphorata were isolated in order to evaluate their selective cytotoxicity toward 14 types of human cancer cell and two non-transformed cell types. Among these triterpenoids, methyl antcinate A (MAA) exhibited the most potent spectrum of anticancer effects in KB cells, four different oral cancer cell lines (TSCCa, GNM, OC-2, and OEC-M1), Panc-1, BT474, PC-3, OVCAR-3, HeLa, and U2OS cells with high selectivity indices (CC(50)/IC(50)). The expression of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and poly(ADP-ribose) polymerase (PARP) of PC-3 cells tested by western blotting suggested that MAA exerts cell death through the caspase-dependent cascade and the Bax-mediated mitochondrial apoptotic pathway, not only on liver and oral cancer cells but on other types as well, including prostate cancer, in a dose-dependent manner. In addition to MAA, methyl antcinate B, dehydroeburicoic acid, and 15α-acetyl-dehydrosulfurenic acid also exhibited significant selective cytotoxic effects to respective cancer cells. Modifications of these triterpenoids may lead to the development of more potent anticancer drugs.

In vitro and in vivo anticancer effects of destruxin B on human colorectal cancer.

AIM: The study of the anticancer effects of destruxin B (DB) is rare and its anticancer mechanism remains unknown. The aim of this study was to test the in vitro and in vivo anticancer effects of DB, on human HT-29 colorectal cancer (CRC). MATERIALS AND METHODS: DB was isolated and characterized by high pressure liquid chromatography, electrospray ionization mass spectrometry and (1)H-nuclear magnetic resonance spectroscopy. (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess the effects of DB on HT-29 cells in vitro. The anticancer effects of DB were investigated in a murine xenograft model of human colon cancer. RESULTS: A significant inhibition of cell viability was observed with DB treatment in time- and dose-dependent manners. DB administered subcutaneously daily at 0.6-15 mg/kg was proven to be safe and effective in inhibiting the growth of CRC cells. Expression of Bax, cleaved poly (ADP-ribose) polymerase and active caspase-3 were observed with DB treatment and the increase in tumor volumes of treated groups were significantly (p<0.05) lower than those of the mock-treated group. CONCLUSION: DB has potential as a new therapeutic agent against human CRC.

Association of estrogen receptor α gene PvuII and XbaI polymorphisms with non-small cell lung cancer.

Single nucleotide polymorphisms (SNPs) of the estrogen receptor (ER)-α have been found to be associated with various diseases at significantly different frequencies. However, whether any relationship exists between ER-α polymorphisms and lung cancer remains to be determined. In this study, 84 non-smoking, female, non-small cell lung cancer patients with various stages of disease and 234 cancer-free reference controls were enrolled to examine the association of ER-α polymorphisms in lung cancer. Two restriction SNP sites, PvuII and XbaI, in the first intron of the ER-α gene were genotyped by polymerase chain reaction-restriction fragment length polymorphism. The frequencies of the PvuII-XbaI haplotypes and genotypes in a Taiwanese population were revealed for the first time. Although the genotypic frequencies of two polymorphic sites of ER- α were in linkage disequilibrium for the lung cancer group (χ(2)=50.013, d.f.=4) and reference controls (χ(2)=60.797, d.f.=4); and 7 and 8 combined genotypes were present, respectively, the distribution and the major genotypes are different in the two groups (p<0.0001). The p-values for PvuII and XbaI genotypes were significantly different between the lung cancer and reference controls. The PP genotype presence was found to be significantly lower in the lung cancer group (P=0.005), whereas presence of the xx genotype was significantly higher (P=0.042). These findings suggested that the PP genotype had a lower risk of lung cancer; whereas the xx genotype had a higher risk. In comparison with other studies conducted in various populations, it is of note that the pX haplotype frequency of this study was higher than that of other studies, whereas the px haplotype was lower. Moreover, the Xx genotypic frequency of XbaI polymorphisms in the ER-α gene of the reference control group was found to be extremely high, whereas the xx genotypic frequency was extremely low. In conclusion, PvuII-XbaI polymorphisms of the ER-α gene were found to be associated with the risk, but not cancer severity, of non-small cell lung cancer in a Taiwanese population.

In vivo cytokine modulatory effects of cinnamaldehyde, the major constituent of leaf essential oil from Cinnamomum osmophloeum Kaneh.

The purpose of this study was to analyse the major compound in the leaf essential oil of Cinnamomum osmophloeum Kaneh. and to examine its in vivo toxicity and cytokine-modulatory effects. The HS-GC/MS and quantitative HPLC analyses showed the concentrations of the major compounds, cinnamaldehyde, benzaldehyde and 3-phenylpropionaldehyde, in the leaf essential oil of Cinnamomum osmophloeum to be 16.88, 1.28 and 1.70 mg/mL, respectively. Acute and sub-acute toxicity tests identified no significant changes in body weight, liver and kidney function indices, and pathology for the mice treated with up to 1 mL/kg body weight of Cinnamomum osmophloeum leaf essential oil or up to 4 mg/kg body weight of cinnamaldehyde. A murine model was established using ovalbumin (OVA)-primed Balb/C mice treated with various concentrations of Cinnamomum osmophloeum leaf essential oil or cinnamaldehyde daily for 4 weeks. The results of tests with commercial ELISA kits indicated no significant cytokine-modulatory effects in mice treated with Cinnamomum osmophloeum leaf essential oil; however, the serum concentrations of IL-2, IL-4 and IL-10, but not IFN-γ, significantly increased in animals treated with 1 mg/kg body weight of cinnamaldehyde during the 4-week period. The possibility that the other constituents act as antagonists of cinnamaldehyde cannot be excluded.

Methylantcinate A induces tumor specific growth inhibition in oral cancer cells via Bax-mediated mitochondrial apoptotic pathway.

An ergostane type triterpenoid methylantcinate A (MAA) isolated from the fruiting bodies of Antrodia camphorata inhibited the growth of oral cancer cell lines OEC-M1 and OC-2 in a dose-dependent manner, without cytotoxic to normal oral gingival fibroblast cells. The major mechanism of growth inhibition was apoptosis induction, as shown by flow cytometric analysis of annexin V-FITC and propidium iodide staining, caspase-3 activation and DNA fragmentation. The increased expression of pro-apoptotic Bax, poly-(ADP-ribose) polymerase cleavage, and activated caspase-3 and decreased expression of anti-apoptotic Bcl-2 and Bcl-xL were also observed. These results provide the first evidence that the anti-oral cancer effects of MAA may involve a mechanism through the mitochondrial dependent pathway. Thus, results reported here may offer further impulse to the development of MAA analogues as potential chemotherapeutic targets for oral cancer complications.

Possible DNA viral factors of human breast cancer.

Viruses are considered to be one of the high-risk factors closely related to human breast cancer. However, different studies of viruses in breast cancer present conflicting results and some of these works remain in dispute. DNA viruses, such as specific types of human papillomaviruses (HPV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), herpes simplex virus (HSV), and human herpes virus type 8 (HHV-8), have emerged as causal factors of some human cancers. These respective exogenous viruses and the possibility of multiple viral factors are discussed in this review.

Apoptotic death mode of mitomycin C-treated HeLa cells and cellular localization of mitomycin C-induced P-glycoprotein.

Mitomycin C (MMC) is an active antineoplastic agent and is suggested to induce apoptosis in a caspase- dependent manner in human gastric, bladder, and breast cancer cells. In this study, the death mode of human cervical cancer cells (HeLa) induced by MMC and the cellular localization of MMC-induced P-glycoprotein (P-gp) were investigated. The results of caspase-3 activity, Annexin V binding, and DNA fragmentation suggested that the degree of caspase-dependent apoptosis induced by MMC was in a dose-, but not time-dependent, manner. Further, in low-dose (0.0299 microM) and long-term (2 months) treatment with MMC, P-gp is itself extruded from the cells and colocalized with nuclear DNA and the overexpression was achieved.

Myasthenia gravis with thymoma and coexistent central hypothyroidism.

Myasthenia gravis (MG) is a well-known acquired autoimmune neuromuscular disorder. Patients with MG have a higher incidence of autoimmune disease than the normal population. MG is frequently associated with autoimmune thyroid disease, the most common of which is thyrotoxicosis. Associated hypothyroidism is not common, and the central (pituitary) origin, to our knowledge, has not yet been reported. We report an MG patient with thymoma that coexisted with central hypothyroidism, the correction of which is mandatory and significant to achieve remission.

The immunopharmaceutical effects and mechanisms of herb medicine.

In recent years, studies on evaluation of the therapeutic and toxic activity of herbal medicinal products became available and popular. The advances in modern biotechnology have led to discovery of many new active constituents. However, it is a constant challenge to establish the pharmacological basis for efficacy and safety of herbal medicinal products. A better understanding of the effects and bioavailability of phytopharmaceuticals can help in discovering suitable and rational therapies. In this review, we present the bioavailability studies in immune system that has been conducted for some of the more important or widely used phytopharmaceuticals. Furthermore, various new drug targets worthy of using for drug development in immunomodulating herbal medicine area and their regulatory mechanisms are also discussed. Adverse effects, drug interactions, and contraindications are also discussed which show that caution should be exercised when combining phytopharmaceuticals with chemically derived pharmaceutical components.

Relationship between viral factors, axillary lymph node status and survival in breast cancer.

PURPOSE: Our previous study based on the results of polymerase chain reaction and Southern hybridization for the detection of Human papilloma virus (HPV), Cytomegalovirus (CMV), Epstein-Barr virus (EBV), Herpes simplex virus (HSV)-1, HSV-2, and Human herpesvirus (HHV)-8 DNA in non-familial breast cancer patients suggest that the viruses associated with breast cancer are HHV-8 > EBV (P < 0.01). Therefore, efforts were made to further investigate the association between breast cancer with nodal status and viral infections. METHODS: Sixty-two breast cancer patients and their mammary specimens were enrolled in this retrospective study. The presence of these six potential oncogenic viruses was analyzed to establish the relationship between nodal status and treatment outcome. Statistical analyses were used for the assessment of variables, including viral positivity and clinical feature. RESULTS: Viral positivity was not significantly different comparing node-positive and node-negative patients (P > 0.05). When the viral factors were not entered for statistical analyses, no variable was significantly related to overall survival. However, tumor stage, tumor size, nodal status , and estrogen receptor were significantly related to relapse-free survival (P < 0.05). For viral factors, the number of infecting viruses is related to the overall and relapse-free survivals. Only when V0 or V(0, 1) was grouped for comparison with other multiply virus-infected subgroups, were the overall and relapse-free survivals significantly different (P < 0.005 or P < 0.001). The results suggest that HSV-1, HHV-8, EBV, CMV, and HPV were related to overall survival, however, only HHV-8 and CMV were related to relapse-free survival (P < 0.05 or P < 0.01). CONCLUSION: Virus factor is significantly related to human breast cancer, not only in terms of the oncogenetic process, but also in overall and relapse-free survivals.

Young stroke, cardiac myxoma, and multiple emboli: a case report and literature review.

Cardiac myxoma is a source of emboli to the vascular tree, especially to the central nervous system. Although it is rare, its early recognition is particularly important because of its unique clinical features of subsequently leading to intracerebral or subarachnoid hemorrhage, even brain metastases, and its potential for surgical cure. Missing the diagnosis may lead to devastating results, including stroke, even sudden death. A 40-year-old male with no other conventional vascular risk factors such as hypertension, diabetes or hyperlipidemia presented with right hemiplegia, global aphasia, vomiting, and fever. Infarction over the left middle cerebral artery was disclosed on magnetic resonance imaging study, and echocardiogram showed a huge mass, about 5cm in size, on the mitral valve which was histopathologically proved to be a cardiac myxoma. He also presented with multiple emboli to the kidneys and the left eye. There is uncertainty about the role of anticoagulation. The treatment of choice remains surgical excision of the cardiac myxoma which may lead to normalization of serum interleukin-6 levels and resolution of constitutional symptoms, and the intracranial aneurysms may regress and resolve.

The immune response induced by hepatitis B virus principal antigens.

Hepatitis B virus (HBV) infection occurs primarily in hepatocytes in the liver with release of infectious virions and non-infectious empty surface antigen particles into the bloodstream. HBV replication is non-cytopathic. Transient infections run a course of several months, and chronic infections are often life-long. Chronic infections can lead to liver failure with cirrhosis and hepatocellular carcinoma. It is generally accepted that neutralizing anti-HBs antibodies plays a key role in recovery from HBV infection by containing the spread of infection in the infected host and facilitating the removal and destruction of viral particles. However, the immune response initiated by the T-cell response to viral antigens is also important for viral clearance and disease pathogenesis in HBV infection. The three structural forms of the viral proteins, the HBsAg, the particulate HBcAg, and the nonparticulate HBeAg, may preferentially elicit different Th cell subsets. The different IgG subclass profiles of anti-HBs, anti-HBc, and anti-HBe in different HBV infection status were revealed. Moreover, the different IgG subclass profiles in chronic carriers did not change with different ALT and AST levels and may reflect the difference between stimulating antigens, immune response, and the stages of viral disease and provide the basis for the use of vaccines and prophylactic treatments for individuals at high risk of human HBV infection. This review elucidates the detailed understanding of the immune responses induced during transient and persistent infection, and the development of immunotherapy and immunodiagnosis in patients with HBV infection, and possible means of reducing the liver damage.

The study of IgG subclass profiles of anti-hbc in populations with different status of HBV infection.

To study IgG subclasses for the hepatitis B virus (HBV) core antigen (anti-HBc) in different populations, a comparison was made between 104 chronic carriers (60 male and 44 female) and 434 recovered individuals (247 male and 192 female). Biochemistry analyses of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) were also performed. Among the 104 chronic carriers, 21 patients were found to be ALT and AST abnormal (> 25 IU/ml). After comparing these ALT and AST abnormal patients with other ALT and AST normal chronic carriers, no statistical difference was observed in the OD values of the anti-HBe (p > 0.05). The ELISA results showed the anti-HBc IgG subclass pattern was IgG1 > IgG3 > IgG4 in chronic carriers and IgG3 > IgG1 > IgG4 in recovered individuals (p < 0.05). This result suggests the IgG1/IgG3 ratio may be related with HBV status. However, in spite of the different anti-HBc IgG1/IgG3 patterns demonstrated in different populations, both anti-HBc IgG1 and IgG3 concentrations were significantly higher in chronic carriers (p < 0.05). Therefore, both the anti-HBc IgG1/IgG3 ratio and their amounts differed. They may play a significant role in chronic carriers and recovered individuals. The anti-HBc IgG subclass profiles of chronic carriers were not changed regardless of liver inflammation, and were independent of sex and age.

Study of the viral infections and cytokines associated with recurrent aphthous ulceration.

Mouth ulcers are one of the most common oral complaints. However, the association between oral ulceration and viruses and cytokines is uncertain. We detected the presence of human papilloma virus (HPV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV)-1, HSV-2 and human herpesvirus (HHV)-8 DNA in oral tissues by polymerase chain reaction (PCR) and Southern hybridization techniques, and quantified the serum levels of cytokines including interleukin (IL)-2, IL-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), soluble Fas (sFas) and the Fas ligand (FasL) by enzyme-linked immunosorbent assay (ELISA) for 67 recurrent aphthous ulcer (RAU) patients and 72 normal individuals. Seven patient specimens were excluded from the study due to the negative PCR results for the beta-globin used as the internal control. Among the 32 (53.3%) virus-positive results from 60 patients' samples, 8 (13.3%) HPV, 4 (6.7%) HSV-1, 11 (18.3%) CMV, 9 (15.0%) EBV, and 16 (26.7%) HHV-8 samples proved to be positive. No HSV-2-positive samples were found. The percentage of single-virus infection (56.3%) was significantly greater than that of double-virus co-infection (31.3%) and the percentage of double-virus co-infection was significantly greater than the percentage of triple-virus co-infection (12.5%) (P < 0.05). In the 72 normal oral-tissue specimens, no viral DNA was detected. The mean serum cytokine level for patients was significantly (P < 0.05) greater than for controls for most of the separate age groups. The mean serum cytokine concentrations for the patient group demonstrated a diffuse pattern covering a wide range of serum concentrations, a very different result from the compact serum concentration pattern and lower mean serum cytokine concentrations revealed by the normal group. Overall association between viruses and recurrent aphthous ulceration is HHV-8 > CMV > EBV > HPV > HSV-1, regarding the frequency of prevalence (P < 0.05).

Inflammatory cytokines reaction elicited by root-end filling materials.

Cytokines are inflammation in the list of tissue reactions that cytokines control of cell and tissue growth, development, and differentiation. Root-end filling materials often contact with existing periapical tissue, and they need to be biocompatible with remnant periapical tissue. The aim of this study was to focus the effects of the root end filling materials on bone cell viability and expression of inflammatory cytokines and their role in maintaining health and stability of the restored dental tissues. Calcium hydroxide-based (Life), zinc oxide eugenol-based (Super EBA), and mineral trioxide aggregate-based (MTA) root-end filling materials were used to investigate their effect on a human osteosarcoma cell line (U2OS). The cell attachment assay was observed microscopically, and the expression of interleukin-2, -4, and -10 were quantified by enzyme-linked immunosorbent assay. Any resultant difference between the root-end filling material was analyzed statistically by one-way analysis of variance. The results showed that the best cell attachment to root-end filling material occurred with MTA. The IL-4 (0.824 +/- 0.396) and IL-10 (2.06 +/- 1.24) levels were greater for the MTA group, whereas IL-2 expression for the three kinds of root-end filling materials was similar. All materials were able to induce expression of inflammatory cytokines from cultured bone cells.

Biocompatibility of human osteosarcoma cells to root end filling materials.

Ideal root end filling materials should have good physical and chemical properties, and the most important is that the material should be biocompatible with periradicular tissue. The biocompatibility of three root end filling materials, mineral trioxide aggregate, calcium hydroxide-based cement, and eugenol-based cement, were investigated in vitro by culturing extracts of these materials with human osteogenic sarcoma cells (U2OS). Extracts of each of the materials were made after incubation of the materials for 1 day and 1 week with complete McCoy's medium. The extracts were serially diluted and then incubated with U2OS cells for 24 and 48 h. Cell survival rates were assessed by means of a viability assay for mitochondrial dehydrogenase activity. Differences in mean cell survival rates were statistically assessed using one-way ANOVA. Results showed that the survival rates of U2OS cells were largest with mineral trioxide aggregate, followed by calcium hydroxide-based cement and eugenol-based cement at 24- and 48-h exposures using the 1-day and 1-week extracts. The duration of root end filling material extraction time and treatment time showed variable influence on the survival rates. The results suggest that mineral trioxide aggregate is more biocompatible than the other root end filling materials and is suitable for use in the clinical setting.

Association of viral factors with non-familial breast cancer in Taiwan by comparison with non-cancerous, fibroadenoma, and thyroid tumor tissues.

To study the etiologic factors of non-familial breast cancer, the polymerase chain reaction (PCR) and Southern hybridization were used to detect six viruses including human papillomavirus (HPV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV)-1, HSV-2, and human herpesvirus (HHV)-8 DNA in 69 patients with breast cancer and 60 specimens from non-cancerous or other individuals with thyroid tumors or fibroadenoma (non-breast cancer controls). Two specimens from patients with a familial history of breast cancer and five breast cancer specimens with negative results for beta-globin, which was used as internal control, were excluded from this study. Eight (12.9%) HSV-1, 28 (45.2%) EBV, 47 (75.8%) CMV, 8 (12.9%) HPV, and 28 (45.2%) HHV-8 positive samples out of the 62 breast cancer specimens were detected; no HSV-2 DNA was detected in any group. Among the viral gene-positive breast cancer samples, 12 (23.1%) were positive for 1 virus, 16 (30.8%) were positive for 2 viruses, 21 (40.4%) were positive for 3 viruses, and 3 (5.8%) were positive for 4 viruses. Among the viral gene-positive specimens of the control groups, only one virus, CMV, was found in the non-cancerous and thyroid tumor specimens, while multiple viruses were found in the fibroadenoma specimens. The viruses associated with breast cancer were HHV-8 > EBV (P <0.01). The viruses associated with fibroadenoma were HSV-1 and HHV-8 > EBV (P <0.01). The presence of more than one virus was found predominantly in breast cancer and exclusively found in fibroadenoma. CMV was the only virus associated with thyroid tumors.