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Human Papilloma virus
Human papillomaviruses (HPV) are common viruses that can cause warts. There are more than 100 types of HPV. Most are harmless, but about 30 types put you at risk for cancer. More than 40 HPV types can be sexually transmitted, and these HPVs spread very easily through genital contact. These types affect the genitals and you get them through sexual contact with an infected partner. They are classified as either low-risk or high-risk. Low-risk HPV can cause genital warts. High-risk HPV can lead to cancers of the cervix, vulva, vagina, and anus in women. In men, it can lead to cancers of the anus and penis.
(http://www.nlm.nih.gov/medlineplus/hpv.html), (Division of STD prevention report, 1999)
The Food and Drug Administration has approved two vaccines, Gardasil and Cervarix, that are highly effective in preventing persistent infections with the two HPV types that cause most cervical and anal cancers. Gardasil also prevents infection with the two HPV types that cause most genital warts.
2.1.1 Human papilloma virus and its structural properties
HPVs cause a diverse range of epithelial lesions. Over 100 different HPV types have been identi¬ed based on DNA sequence analysis (Bernard, 2005), with each being associated with infection at speci¬c epithelial sites (Villiers de, 2001). At an evolutionary level, HPVs fall into a number of distinct groups or genera and the lesions they cause have different characteristics. The two main HPV genera are the Alpha and Beta papillomaviruses, with approx. 90% of currently characterized HPVs belonging to one or other of these groups. Beta papillomaviruses are typically associated with in apparent cutaneous infections in humans but, in immunocompromised individuals and in patients suffering from the inherited disease EV (epidermodys plasia verruciformis), these viruses can spread unchecked and become associated with the development of non- melanoma skin cancer (Harwood et al., 2002, P¬ster 2003).
2.1.2 Virus types and its role in cancers
The Human papilloma virus and their subtypes are associated with cutaneous mucosal and anogenital diseases (Table 2.1.1). HPV DNA probably found in 99.7% of all cervical carcinomas, with HPV types 16, 18, 45 and 31 being the most frequent. (Walboomers et al., 1999, Koutsky et al., 2002) Based on these observations, the anogenital HPVs have been divided into two groups: the first is associated with a high risk for cervical cancer development - the HR HPVs (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82), and the second group with a low carcinogenic potential - the low-risk (LR) HPVs (6, 11, 40, 42, 43, 44, 54, 61, 72 and 81)(Munoz et al, 2003). It has now been proven beyond reasonable doubt that infection with an HR HPV is a necessary prerequisite for the development of cervical cancer, and the World Health Organization (WHO) has recognized HPV 16 and HPV 18 as carcinogenic agents for humans.
More than 90 types of HPV (pathogenic and non-pathogenic) have been identified. The most common ones associated with cervical cancer are types 16, and 18, which have been reported in 70% of the cases. The implication of the importance of pathological human Papillomavirus has not been studied in the Middle East where the incidence of cancer of cervix is high (Mortazavi et al., 2000).
Persistent HPV infections are now recognized as the cause of essentially all cervical cancers, as well as most cases of anal cancer. In 2011, more than 12,000 women in the United States are expected to be diagnosed with cervical cancer and more than 4,000 are expected to die from it. (American Cancer Society, Cancer Facts and Figures 2011). Cervical cancer is diagnosed in nearly half a million women each year worldwide, claiming a quarter of a million lives annually.
Although anal cancer is uncommon, more than 5,000 men and women in the United States are expected to be diagnosed with the disease in 2011, and 770 people are expected die from it.
Genital HPV infection also causes some cancers of the vulva, vagina, and penis (Parkin, 2002). In addition, oral HPV infection causes some cancers of the oropharynx (the middle part of the throat, including the soft palate, the base of the tongue, and the tonsils) (Parkin, 2002, D'Souza et al., 2007).
It has been estimated that HPV infection accounts for approximately 5 percent of all cancers worldwide (Parkin, 2002).
Nongenital Cutaneous Disease
Common warts (verrucae vulgaris)
1, 2, 4, 26, 27, 29, 41, 57, 65
Plantar warts (myrmecias)
1, 2, 4, 63
Flat warts (verrucae plana)
3, 10, 27, 28, 38, 41, 49
Butcher's warts (common warts of people who handle meat, poultry, and fish)
1, 2, 3, 4, 7, 10, 28
2, 27, 57
Ungual squamous cell carcinoma
Epidermodysplasia verruciformis (benign)
2, 3, 10, 12, 15, 19, 36, 46, 47, 50
Epidermodysplasia verruciformis (malignant or benign)
5, 8, 9, 10, 14, 17, 20, 21, 22, 23, 24, 25, 37, 38
Nonwarty skin lesions
Nongenital Mucosal Disease
Squamous cell carcinoma of the lung
6, 11, 16, 18
6, 11, 30
Maxillary sinus papilloma
Squamous cell carcinoma of the sinuses
Oral focal epithelial hyperplasia (Heck disease)
Squamous cell carcinoma of the esophagus
6, 11, 30, 42, 43, 44, 45, 51, 52, 54
16, 18, 34, 39, 42, 45
16, 18, 31, 34
Giant condylomata (Buschke-Löwenstein tumors)
Unspecified intraepithelial neoplasia
30, 34, 39, 40, 53, 57, 59, 61, 62, 64, 66, 67, 68, 69
Low-grade intraepithelial neoplasia
6, 11, 43
Intermediate intraepithelial neoplasia
31, 33, 35, 42, 44, 45, 51, 52
High-grade intraepithelial neoplasia
16, 18, 56, 58
Carcinoma of vulva
6, 11, 16, 18
Carcinoma of vagina
Carcinoma of cervix
16, 18, 31
Carcinoma of anus
16, 31, 32, 33
Carcinoma in situ of penis (erythroplasia of Queyrat)
Carcinoma of penis
16, 18Table 2.1.1: Diseases and Associated HPV Subtypes
2.2 Human Papilloma virus Pathogenesis
2.2.1 Pathogenesis of virus in cancers
Papillomaviruses have icosahedral symmetry and are non-enveloped. Seventy-two capsomeres surround the genome. A major and minor capsid protein comprises the outer protein coat of the virus. Approximately 8000 base pairs comprise the closed, double stranded- circular HPV genome.
Fig. 2.1: Chart I - HPV Gene Coding Regions
Coding information for HPV exists on one strand; it is believed that transcription occurs in a clockwise direction from a single promoter region (P97). Three major regions comprise the HPV genome. The early region (E1-8) consists of genes responsible for transcription, plasmid replication, and transformation. The late region codes for the major (L1) and minor (L2) capsid proteins and the control region contains the regulatory elements for transcription and replication.
HPV infection occurs at the basal epithelium. Although the incidence of infection is high, most infections resolve spontaneously. A small proportion of infected persons become persistently infected; persistent infection is the most important risk factor for the development of cervical cancer precursor lesions.
The most common clinically significant manifestation of persistent genital HPV infection is cervical intraepithelial neoplasia, or CIN. Within a few years of infection, low-grade CIN called CIN-1 may develop, which may spontaneously resolve and the infection clear.
Fig. 2.2 Natural History of HPV Infection
Persistent HPV infection, however, may progress directly to high-grade CIN, called CIN2 or CIN3. High-grade abnormalities are at risk of progression to cancer and so are considered cancer precursors. A small proportion of high-grade abnormalities spontaneously regress. If left undetected and untreated, years or decades later CIN2 or 3 can progress to cervical cancer.
Infection with one type of HPV does not prevent infection with another type. Of persons infected with mucosal HPV 5% to 30% are infected with multiple types of the virus.
It is believed that the HPV virus enters the body after slight trauma to the epithelium and needs terminally differentiated epithelial cells for replication. Late-region genes are expressed in the differentiated cells near the epithelium's surface providing an easy mode of viral transmission. Early region genes are expressed in the basal cells of the epithelium but are unable to produce virus since these cells do not make the capsid proteins encoded by the late genes.
All types of HPV replicate only within the host cell's nucleus, but the mechanism by which HPV types transform cells is unclear. Most studies focus on HPV-16 and HPV-18, the viruses most frequently associated with anogenital carcinomas. The HPV genome replicates as an extra chromosomal episome or plasmid in benign HPV-associated lesions. However, the viral DNA is often integrated into the host's chromosome in malignant HPV associated lesions. E6 and E7 genes are strongly associated HPV-mediated carcinogenesis.
Another study indicating a positive association between HPV 16 and high grade lesions, suggests that additional cofactors, such as cigarette smoking, may be required as a carcinogen to advance HPV-infected cells toward neoplastic progression. All HPVs infect the mucous membranes of the skin. However, the various forms of HPVs will establish themselves in distinct cell types. Thus, HPVs are divided into three categories: genital mucosal and nongenital cutaneous types and types specific for epidermodysplais verruciformis. Moreover, people who have visible genital warts can be infected with multiple HPV types concurrently.
Most HPV infections are asymptomatic. However lesions can develop anywhere in a time frame between three weeks and eight months after infection, with most developing 3 months after infection. Of the HPVs characterized with an oncogenic effect, it is shown that carcinoma rarely develops immediately after infection.
HPV-6 and HPV-11 are most commonly associated with genital warts. While the most common presentation is warts, or condylomata, many infections are detected only by Pap smear cytological evidence. In contrast to the strong association between cervical intraepithelial neoplasia and HPV, the relationship between HPV and squamous epithelial lesions is less clear. One study's findings support the hypothesis that HPV may be associated in the development of ovarian squamous intraepithelial neoplasia.
2.2.2 Pathogenesis of human papilloma virus in oral cancers
Human papillomaviruses infection is known to be a necessary element for the development of cervical cancer in women and is also a risk factor for the development of anal, penile, and vulvar cancers. The site that is mostly associated with HPV infection in the head and neck area is the oropharynx, particularly the tonsils and tongue base. It is not clear why the oropharynx is more susceptible to HPV transformation.
The mechanism of Human papillomaviruses carcinogenesis was first characterized in cervical cancer, where more than 90% of cases are related to HPV infection. Human papillomaviruses have been categorized by their genotypes into low-risk and high-risk types according to the risk of that virus causing squamous cell carcinoma of the uterine cervix (Steben et al., 2007). Infection of the uterine cervix with any human papillomavirus (HPV) genotype is associated with high-risk sexual behaviour, particularly if started at a younger age; and persistent infection of the uterine cervix with high-risk HPV genotypes, especially HPV-16 and HPV-18, is essential for the development of squamous cell carcinoma (SCC) (Walboomers et al., 1999, Monk et al., 2007).
Recent evidence also incriminates high-risk HPV-genotypes in the pathogenesis of oral and oropharyngeal SCC (Miller et al., 2001, Mork et al., 2001, Syrjanen et al., 2003, Gillison et al., 2004, Syrjanen S , 2005, Licitra et al., 2006, D'Souza et al., 2007, Haddad RI, 2008). HPV infection of the mouth and of the oropharynx like HPV infection of the uterine cervix, is associated with high-risk sexual behaviour, in particular with oro genital sex; and high-risk HPV genotypes, in particular HPV-16, are present in many oral and oropharyngeal SCC where in some cases they probably play an essential aetiological role(D'Souza et al., 2007). Persons with oropharyngeal SCC in which HPV can be detected intracellularly have a better prognosis than persons with HPV-cytonegative oropharyngeal SCC (Gillison et al, 2000, Licitra et al ., 2006).
The demonstration of HPV DNA, even of high-risk HPV oncogenes in squamous cell carcinoma is not in itself sufficient evidence of oncogenesis by the HPV in that context. HPV may well have been either present but a non-participant during the oncogenesis, or have been superimposed upon the malignancy. On the other hand, absence of HPV DNA from any carcinoma does not exclude the theoretical possibility of its having played some role in the initiation of the malignancy since HPV infections are frequently transient(Mork et al, 2006). In such a 'hit and run' situation, HPV may incite initial transformation in cells that subsequently lose their HPV DNA sequences during carcinogenesis (Syrjanen, 2003). However, this is highly improbable since persistence of oncoproteins E6, E7 of the high-risk HPV genotypes appears to be necessary for the perpetuation of HPV-associated malignancy, as is evident from the presence of HPV DNA in the cells of SSC of the uterine cervix.
Acquisition of oral and oropharyngeal HPV infection
Both oral and oropharyngeal HPV infection and oral and oropharyngeal SCC are associated with the practice of orogenital sex and with the high-risk sexual behaviour of cohabiting with many partners, particularly when started at a younger age (Mork et al., 2001, Gilson et al., 2004, Syrjanen., 2007, D'Souza., 2007, Kreimer et al., 2004). In a study primarily aimed at vulvogenital HPV infection, tobacco smoking and increasing age were found to be risk factors associated with increased frequency of persistent oral HPV infections in women. This appears to be because tobacco-mediated and age-related local genetic and immune deregulation renders the tissues more susceptible to HPV infection (D'Souza et al., 2007).
Although oral and oropharyngeal HPV infections are primarily sexually acquired, mouth to mouth contact between partners and between family members, autoinoculation, and vertical birth-transmission are also routes whereby HPV infection of oral and oropharyngeal sites can be established (Fakhry et al., 2006). As oral and oropharyngeal subclinical HPV infection is not uncommon, it is possible that the epithelium may serve as a reservoir of the virus and when activated the virus may play a role in HPV-associated oral and oropharyngeal SCC.
HPV in oral and oropharyngeal squamous cell carcinoma
The HPV E6 and E7 oncogenes, which encode proteins consisting of 151 and 98 amino acids, respectively, are largely responsible for the onset and persistence of the malignant process in both anogenital and head and neck cancers. E6 and E7 are best known for their ability to bind and inactivate the tumor suppressors p53 and pRb, and these respective properties have been associated with their oncogenic potential. The E6 protein contains zinc-binding motif and can form a complex with the p53 tumor suppressor protein of the host cell, inducing p53 degradation. The E7 protein forms complexes with retinoblastoma gene family proteins, which are negative regulators of cell growth. This results in the release of the E2F transcription factors in the cell. The free E2F activates the expression of several host genes involved in the cell cycle progression, and the E6/E7-inactivated p53 and pRb-related proteins permit the cell to escape normal check points, with subsequent loss of DNA replication. The simultaneous effects of loss of both p53 and pRb function may lead to the malignant transformation of epithelial cells.
This absence of genetic or epigenetic alterations in the p53 and pRb pathways in HPV-positive SCCHN is in sharp contrast to what is observed in HPV-negative disease. Typically, p53 mutations are frequently observed in the HPV-negative squamous cell carcinomas. By contrast, HPV-positive carcinomas usually do not contain any p53 mutations, and occur predominantly in patients with no excessive tobacco and/or alcohol consumption history. These distinctions imply that HPV-positive and HPV-negative carcinomas of the head and neck represent distinct entities. Moreover, it has been suggested that the prognosis of patients with HPV-positive tumors is better than that of patients with a smoking related, HPV negative tumor. The association between HPV infection and other tumor suppressor genes, such as p16, is also of interest. The p16 protein functions as a tumor suppressor by binding to the cyclin D1 CDK4/CDK6 complex, preventing phosphorylation of the Rb protein. Overexpression of p16 protein has been reported repeatedly in HPV-associated cancers, and one of study, only the HPV-positive/p16 expressing tumors close to 25% of tumors analyzed were the ones associated with favorable prognosis.
In epidemiological studies,SCC of the head and neck is frequently treated as a homogeneous group, and the various component carcinomata (oral, oropharyngeal, laryngeal, nasopharyngeal, hypopharyngeal etc.) are not often separated out statistically. The reported rates of detection of HPV DNA in head and neck SCC range from 0 to 100% (Syrjanen et al., 2007, Ha et al., 2004]. This extreme variation in reported prevalence may be owing to lumping together of essentially different lesions; to small sample numbers; and to differences in the sampling techniques; in the ethno-geographic origins of the subjects examined; and in the HPV detection methods applied.
2.2.3 Pathogenesis of human papilloma virus in cervical cancers
As long ago as 1995 the causative association between HPV and SCC was recognised (International Agency for Research on Cancer, 1995). The HPV family of viruses contains more than 100 types; some HPVs cause benign skin warts, or papillomas, for which the virus family is named. Approximately 40 HPV types affect the genital area. They can be sub-divided into those that are low risk for cervical cancer (such as HPV-6 and HPV-11, which are responsible for causing genital warts) and those which are high risk for cervical cancer. The high risk types occurring most frequently in cervical cancer include HPV-16 and HPV-18; together these account for over 70% of SCCs (Munoz, 2004). HPV-18 is also thought to account for approximately 50% of all adenocarcinomas.
At least 15 human papillomavirus (HPV) types are human carcinogens that play a central role in the pathogenesis of cervical cancer and other less common cancers, including vaginal, vulvar, anal, penile, and upper aerodigestive tract cancers (Munoz N et al., 2003, Gillison ML et al., 2003) Worldwide, cervical cancer is the second most common cancer in women, with the majority of cases (~80%) occurring in developing countries (Parkin DM et al., 2001). In the US and other developed countries, cervical cancer is no longer a leading cause of cancer-related mortality (Rieslag et al., 2002) because women undergo routine Pap screening and are treated if cervical intraepithelial neoplasia (CIN) is diagnosed. CIN, however, continues to cause significant psychological and physical morbidity.
The association between certain strains of HPV and cervical cancer is well known. In the West, cervical cancer is the fourth most common cancer in women. In developing countries though, it is the most common cancer, with 1/2 million new cases per year. Overall, it is the most common virus associated cancer worldwide, followed by hepatocellular carcinoma, NPC and Burkitt's lymphoma. There is now compelling evidence for an aetiological role of HPV in cervical and other cancers.
A) Epidemiological Evidence:
It has been known for a long time that the incidence of cervical Cancer. is related to sexual activity. Promiscuous women are far more likely to develop cervical cancer. Nuns rarely get cervical cancer. Several sexually transmitted agents have been implicated including Herpes Simplex. It is now fair to say that evidence of a role for HSV is very slim and now very much discounted.
B) Virological Evidence:
It has now been proven that certain precancerous conditions for cervical and vulval cancer, namely CIN and VIN, are caused by HPV. Normal human epithelium from the cervix and epithelium purposefully exposed to HPV 16 were transplanted to nude mice. (Immunologically immature mice which are not able to reject the graft). Weeks later, lesions that resemble CIN histologically were detected in the epithelium which was infected by HPV.
HPV genomes can be detected in 90 - 95% of tumor cell lines. HPV genomes can be found at a lower frequency in pre cancerous state such as CIN1, CIN2 and CIN3. HPV 16 and 18 are the most commonly found. However, other strains may be present, especially in the less dysplastic lesions. Types 6 and 11 are commonly present in the less dysplastic lesions but not in the more dysplastic ones.
HPV DNA persists in an episomal form in normal infected cells but is integrated in the host cell chromosome in malignant cells. The integration of HPV DNA appears to be random. It is thought that as integration occurs, the breakage always occurs in the E1 region. One of the functions of the E1 region is to negatively control the expression of the E6 and E6 genes. Therefore, it is thought that as a result of the integration, the E1 function is lost and the E6 and E7 genes are derepressed. This leads to an overproduction of the E6 and E7 gene products. E6 is shown to inactivate p53 and E7 the Rb proteins.
HPV DNA is biologically active in vitro. It is able to transform human fibroblasts and keratinocytes. It has effects on epithelial cell differentiation and induces DNA replication in the fibroblast. All the above effects can be nailed down to the E7 reading frame. The constant expression of the E7 and perhaps the E6 reading frames products are essential for the maintenance of transformation.
Antibodies to HPV are more prevalent in patients with CIN or invasive Ca. Gissman et al. looked at the prevalence of antibodies to the E7 and E4 HPV proteins in normal people and those with cervix cancer . Although the prevalence of Abs to E4 and E7 were increased in patients with Cervix cancer compared to the controls. The prevalence of Ab to E7 was much higher; There was only a 2 fold difference in the prevalence of Ab to E4 between the cancer patients and the normal controls. However there was a 7-fold difference in the prevalence of Ab to E7. This suggests that there may be a role for testing for Ab to the E7 protein in future for use as a marker in monitoring the effectiveness of treatment. However, it is very important to have the correct protein antigen of the particular strain of HPV involved.
It must be born in mind that other factors are involved in the tumourigenesis of cervical cancer
The actual site in the body
The HPV type
The immune status of the patient; immunocompromised patients have a much higher risk of developing cervix cancer.
Vitamin A deficiency
3. Association with tumours in immunocompromised individuals
10% of the general population have warts at any one time but warts and skin cancers are much more common and of a greater problem in immunocompromised patients, in particular renal transplants and people with AIDs. The actual numbers of people undergoing renal transplant operations have increased gradually in the last 20 years. As survival times increased, it became apparent that many of them are developing serious problems with warts and skin cancers. The problem seems to be related to the maintenance immunosuppressive therapy, which is prescribed to patients as long-term therapy after their operation. Prednisolone and Azothiaprine are the 2 most common immunosuppressants given.
Many renal transplant patients appeared with horrific intractable warts in all parts of the body. The warts could not be controlled by any methods but may respond to the withdrawal of the immunosuppressive therapy. Some of these patients with warts progressed onwards to malignancy in many parts of the body at once. Several factors are known to contribute to malignancy:
Oncogenic effects of the drugs given
Chronic antigenic stimulation
The longer the graft survives, the more likely that patient will develop warts and skin cancers.
Table 2.2 Graft survival in different stages
Age of Patients
< 5 years
> 5 years
The widespread appearance of warts in renal transplant patients resembles those found in the condition Epidermaldysplasia Verruciformis (EV). EV is a rare autosomal recessive condition characterized by the appearance of widespread warts on the surface of the body and 30% of the development of squamous cell carcinomas (SCC). Over 20 types of HPV are associated with EV and the HPV DNA exists mainly in the episomal form in the lesions of these patients. In renal graft survivors the ratio of basal cell carcinoma to SCC is 1:15 compared to a ratio of 1:5 for a normal population. The female renal transplant patients are also at a much higher risk of developing cervical Ca., CIN and VIN than the normal population. The precise role of HPV in the causation of skin cancers in the renal-transplant patients is not known. The skin lesions do resemble those of EV on appearance and the DNA of HPV 5 and 8 (the HPV strains commonly isolated from patients with EV) are found in 50% of SCC of the renal transplant patients. HPV 5 and 8 may act as inducing agents for the development of skin tumours. In a recent study, it was claimed that the prevalence of anti-HPV 8 Ab is increased in patients with skin tumours:
Table 2.3: Percentage of prevalence of anti HPV Antibody in patients
Basal Cell Carcinoma
Squamous Cell Carcinoma
If the above can be substantiated, then HPV may have a far more sinister role in human cancers than previously thought. It may have a role in inducing the development of skin and other epithelial cancers. It is not clear whether a withdrawal of immunosuppressive therapy will have a beneficial effect on skin cancers. Although some patients with intractable cancer problems have been offered a withdrawal of immunosuppressive therapy. There have been no cases of metastasis from a SCC arising in an ex-renal transplant patient to date.
4. Persistence of HPV infection
There is circumstantial evidence that HPVs may persist in the squamous epithelium, without producing recognizable lesions, in a fashion similar to the persistence of polyomaviruses in the kidney. Up to 42% of allograft recipients develops cutaneous lesions within a year after transplant. This suggests that transplanted patients experience either new infections or reactivation of latent virus. HPV DNA sequences have been found in biopsies of normal areas of the larynx of individuals who have had recurrent episodes of laryngeal papillomas.
2.2.4 Structure properties of targets
Infection with oncogenic human papillomavirus (HPV), most frequentlyHPV16, is the most signi¬cant risk factor in the aetiology of cervical cancer, as shown by the extremely high frequency (99.7%) of HPV DNA detected in cervical malignancies (Walboomers et al., 1999). Both in vitro and in vivo studies show that the functions of the E6 and E7 proteins of the 'high-risk' HPVs are essential for cellular transformation. The viral oncoproteins E6 and E7 from 'high-risk' HPVs have been shown to alter pathways involved in cell-cycle control, interacting with and neutralizing two important tumour suppressors, p53 and Rb (Vousden et al., 1988). The loss of activity of these two tumour suppressors results in an uncontrolled continuous cellular proliferation leading to cell transformation.HPV16 E6 (16E6) is a small protein composed of 151 or 158 aa, depending on the initiation codon used. 16E6 contains two zinc-binding domains and a C-terminal PDZ-binding domain (Mantovani & Banks, 2001). With the help of the cellular ubiquitinligase E6AP, 16E6 mediates the degradation of p53 (Scheffner et al., 1990). So far, the regions of E6 involved in binding to either p53 or E6AP have mostly been investigated by performing point mutations or deletions of E6 (Cooper et al., 2003; Fosteret al., 1994; Gewin & Galloway, 2001). The E7 protein binds to the hypo phosphorylated form of Rb, resulting in the disruption of Rb/E2F complexes and reversal of the G1/S cell-cycle checkpoint. The E6 and E7 proteins inactivate tumor suppressors p53 and retinoblastoma (Rb) respectively and render the breakdown of cell cycle regulation. Hence, high-risk HPV-infected cells develop genomic instability, which can lead to the progression of cancer.
2.2.5 Genomic organization of targets proteins
Human papillomaviruses (HPVs) belong to the Papillomaviridae family and are small, nonenveloped, icosahedral DNA viruses. The viral particles are 55 to 60 nm in diameter and consist of a single molecule of a double-stranded covalently closed circular DNA genome of approximately 7900 bp. The genomic organization of all papillomaviruses is remarkably similar (Fig 2.3). Viral DNA is associated with cellular histones to form a chromatin-like complex. All coding sequences are located on one DNA strand only. Most papillomaviruses contain six early ORFs and two late ORFs. There is a region with no ORFs which is designated the long control region (LCR), the upstream regulatory region (URR), or the noncoding region (NCR).
Fig.2.3 Genomic organization of HPV-16
ORFs deduced from the DNA sequence are designated E1 to E7, L1, and L2, indicated in grey boxes. A non-coding region (NCR) is indicated by a black box. Main functions of genes are listed.
The HPV E6 and E7 proteins are the only two viral genes expressed in virtually all HPV-positive cervical carcinomas, and many lines of experiments have shown that these are cooperative viral oncoproteins (Munger et al, 2004). The activities of E6 and E7 that are most clearly linked to carcinogenesis are their abilities to inactivate the p53 and the retinoblastoma (pRb) tumor suppressors, respectively. High-risk HPV E6 proteins bind directly to E6AP, a cellular lar ubiquitin ligase encoded by the UBE3A gene, causing its substrate specificity to be altered so that it stably associates with and polyubiquitylates p53, resulting in degradation of p53 by the 26S proteasome.
Therefore E6 acts (at least in part) as an oncoprotein by stimulating the destruction of perhaps the most important tumor suppressor proteins in human cancer. The E7 protein also promotes the proteasome dependent degradation of pRb.
2.3 Material and Methods
2.3.1 Human Papilloma virus and potent targets for oral and cervial cancers
The HPV is a member of the papovaviridae family and contains a double-stranded DNA virus. The papillomaviruses are a diverse group and have been detected in a wide variety of animals as well as in humans. The virus contains a double-stranded, circular DNA genome containing 7800~7900 base pairs, a non enveloped virion, and an icosahedral capsid. Because of the clinical importance, human papillomaviruses have been extensively studied, and at present approximately 118 different subtypes with limited DNA homologies have been identified.
In epidemiological studies, SCC of the head and neck is frequently treated as a homogeneous group, and the various component carcinomata (oral, oropharyngeal, laryngeal, nasopharyngeal, hypopharyngeal etc.) are not often separated out statistically. The reported rates of detection of HPV DNA in head and neck SCC range from 0 to 100% (Syrjanen S., 2007, Ha PK et al, 2004). In a meta analysis of data from 94 studies of a total of 4580 specimens, Miller and Johnston (2001) determined that the prevalence of HPV in normal oral mucosa and in oral SCC is likely to be 10% and 46.5%, respectively (Miller SC et al., 2001).
Serum antibodies against L1, E6 and E7 proteins of HPV-16 were detected in well over 60% of persons with oropharyngeal SCC (D'Souza et al, 2007). Since antibodies to HPV-16 capsid protein L1 are strongly associated with oral and oropharyngeal SCC, and since these antibodies are evidence of long-term exposure to HPV-16, it is possible, indeed probable, that exposure to HPV-16 precedes the development of oropharyngeal SCC by several years (Mork J et al., 2001, Syrjanen S., 2007, D'Souza et al, 2007). However, this observation must be interpreted with caution since other HPV infections, for instance anogenital and oral warts will increase HPV antibody titres, and this can confound the observed association between serum HPV antibody levels and oral and oropharyngeal SCC (Mork J et al., 2001). As is the case with the virus itself, HPV-16 seropositivity is strongly associated with increased risk of developing HPV-cytopositive oropharyngeal SCC, but there is only a weak association for oral SCC (Pintos J et al .,2008, Furnis CS, 2007).
HPV-associated and non HPV-associated (tobacco/alcohol related, idiopathic) oral and oropharyngeal SCC are different in cytogenetic profiles, clinical characteristics and courses of the disease (Gillison et al, 2000, Gillison et al., 2000). While HPV-associated cytopositive oral and oropharyngeal SCC is thought to be initiated and maintained by high-risk HPV E6/E7 oncoprotein-induced dysregulation of cell cycle control mechanisms, leading to genomic instability (Gillison et al.,2000, D'Souza et al, 2007), HPV-cytonegative oral and oropharyngeal SCC often show mutation of p53 tumour-suppressor gene frequent loss of heterozygosity (LoH) at chromosomal loci 3p, 9p and 17p, normal or increased levels of pRb,and decreased levels of p16INK4A [Braakhuis BJ et al ,2004]. HPV-associated and non-HPV-associated pathogenic mechanisms result in distinctly different cellular molecular characteristics (Gillison ML, et al 2004, Haddad RI et al, 2008).
A role for HPV in oropharyngeal tumors is further substantiated by distinct molecular genetic alterations in HPV-positive versus HPV negative tumors. As for many cancers, inactivation of the p53 and pRb pathways is a common event in the molecular progression of HNSCC. However, inactivation occurs by different mechanisms in HPV-positive and -negative tumors. In HPV-positive HNSCC, genetic alterations are re¬‚ective of viral oncogene function. For instance, HPV-positive tumors tend to have wild-type p53, because p53 is functionally inactivated by viral E6 oncoprotein (Gillison ML et al., 2000, Wiest T et al., 2002, Hafkamp HC et al, 2003., Balz V et al, 2003, Haraf DJ et al, 1996 ., Lindel K et al , 2001, 39. Brachman DG et al, 1992, Chiba I et al , 1996). By contrast, HPV-negative tumors have speci¬c p53 mutations demonstrated to be induced by carcinogens in tobacco smoke(Gillison ML et al., 2000, Wiest T et al., 2002, Hafkamp HC et al, 2003., Balz V et al, 2003, Haraf DJ et al, 1996 ., Lindel K et al , 2001, 39. Brachman DG et al, 1992, Chiba I etal, 1996).
As another example, pRb function is inactivated by viral E7 protein in the HPV-positive tumor, but in HPV-negative tumors, the pRb pathway is altered by other mechanisms, including ampli¬cation of cyclin D and inactivation of p16. More complex differences in regions of chromosomal loss and gain have been demonstrated in HPV-positive versus-negative tumors through techniques such as comparative genomic hybridization and microsatellite analysis.
2.3.2 Resources for the ligands preparation:
The following resources were use for the ligands 3 dimensional and PDB ( Protein databank) file format for Insilco target- ligand interactions.
National Center for Biotechnology Information (NCBI): This is one of American government resource for the for molecular biology information, NCBI's mission is to create new information technologies to help in the understanding of fundamental molecular and genetic processes that control health and disease. The NCBI has been stimulating with creating automated systems for storing and analyzing understanding about molecular biology, biochemistry, and genetics; facilitating the use of such databases and software by the research and medical community; coordinating efforts to gather biotechnology information both nationally and internationally; and performing research into advanced methods of computer-based information processing for analyzing the structure and function of biologically important molecules.
Pubchem Compound Database for collection of chemical structure of ligands:
PubChem database was released in 2004. This database provides information on the biological activities of small molecules. PubChem is constructed which governs the three linked databases within the NCBI's Entrez information retrieval system. These are PubChem Substance, PubChem Compound, and PubChem BioAssay. PubChem also provides a fast chemical structure similarity search tool. Information related to use each component database may be found using the links in the homepage of this database through NCBI home page (http://pubchem.ncbi.nlm.nih.gov/).
The links from PubChem's chemical structure records to other Entrez databases provide information on biological properties. These include links to PubMed scientific literature and NCBI'sprotein 3D structure resource in this database. This has been used to obtain the 3-D structures of ligands for the insilico interaction study of compounds.
ChemDraw Pro 13.0 for the ligands structures generation: ChemDraw is the drawing tool of choice for chemists to built publication-ready, scientifically intelligent drawings for use in databases and publications. The tools also can be use for querying chemical databases.
This is important set of tools to handle substructural query types (such as variable points of attachment, ring/chain size, R groups, atom/bond/ring types, and generic atoms) ensures that compounds are quickly and accurately located by searches, no matter how they are stored in commercial, public or in-house databases.
2.4 Results and Discussion
Cancers arise by an evolutionary process as somatic cells mutate and escape the restraints that normally rein in their untoward expansion. Consequently, multiple mechanisms have arisen to forestall uncontrolled cell division. Some of these are devices within the cell, such as those that limit cell-cycle progression, whereas others are social signals that prompt a cell to remain within its supportive microenvironment. In combination, these tumor suppressing mechanisms are remarkably effective and can discriminate between neoplastic (abnormally growing) and normal cellular states and efficiently quell the former without suppressing the latter. It is interesting to note that many, perhaps all, networks that drive cell proliferation harbor intrinsic growth-suppressive properties. Such innate inhibitory functions obscure any immediate selective advantage that mutations in such pathways might otherwise confer. Because no single pathway confers a net growth advantage, any proto-cancer cell acquiring any single oncogenic mutation is effectively trapped in an evolutionary cul-de-sac. By contrast in normal cells, coordinated extra-cellular cues activate multiple pathways in concert. In this way the inherent growth-suppressive activity of each pathway is gated by another, thereby unlocking the cell's proliferative potential. However, deregulation of one or more of these activities may ultimately lead to cancer.
Table-2.5.1 Selected Anticancer Ligands for oral and cervical cancers targets based on ayurveda
No. of Ligands
Ligands with CID
(Compound Identifier No)
Structure of Ligands
Bis Demethoxy curcumin
Curcumin di piporyl ester
Di benzoyl methane
Pdb structure generated
Pdb structure generated
Pdb structure generated
Pdb structure generated
Pdb structure generated
Turmeric has been used for thousands of years in Ayurvedic and traditional Chinese medicine. In modern times, curcumin, the yellow pigment of the spice turmeric, continues to be used as an alternative medicinal agent in many parts of South East Asia for the treatment of common ailments such as stomachic upset, flatulence, jaundice, arthritis, sprains, wounds and skin infections among many others. Curcumin is a component of turmeric; the yellow spice derived from the roots (rhizomes) of the plant Curcuma longa. Curcuma longa is a short-stemmed perennial, which grows to about 100 cm in height. It has curved leaves and oblong, ovate or cylindrical rhizomes. Curcuma longa grows naturally throughout the Indian subcontinent and in tropical countries, particularly South East Asia. A traditional remedy in "Ayurvedic medicine" and ancient Indian healing system that dates back over 5,000 years, turmeric has been used through the ages as an "herbal aspirin" and "herbal cortisone" to relieve discomfort and inflammation associated with an extraordinary spectrum of infectious and autoimmune diseases (Sharma et al., 2005).
Curcumin, chemically it is known as diferuloylmethane (C21H20O6), has been the subject of hundreds of published papers over the past three decades, studying its antioxidant, anti-toxic, anti-inflammatory, cancer chemopreventive and potentially chemotherapeutic properties (Campbell et al., 2005,Sharma et al., 2005). The pharmacology and putative anticancer properties of curcumin have been the subject of several review articles published since 1991, which predate a number of clinical studies of curcumin which have been completed and published within the last few years (Ammon et al., 1991).The anticancer compounds taken for the insilico interaction studies for oral and cervical cancer are listed (Table-2.5.1).