SARS-CoV-2 Infection of Salivary Glands Compromises Oral Antifungal Innate Immunity and Predisposes to Oral Candidiasis.

SARS-CoV-2 targets salivary glands potentially impacting oral health. We show that presence of replicating viruses in the acinar cells of salivary glands compromises production and secretion of histatin-5, a key host-produced antifungal peptide. The salivary levels of histatin-5 were significantly reduced in SARS-CoV-2 infected subjects, concomitant with enhanced prevalence of the fungal opportunistic pathogen Candida albicans. These findings provide direct evidence associating SARS-CoV-2 infection with predisposition to oral candidiasis.

A Longitudinal Metagenomic Comparative Analysis of Oral Microbiome Shifts in Patients Receiving Proton Radiation versus Photon Radiation for Head and Neck Cancer.

Introduction: Due to the radiation-sparing effects on salivary gland acini, changes in the composition of the oral microbiome may be a driver for improved outcomes in patients receiving proton radiation, with potentially worse outcomes in patients exposed to photon radiation therapy. To date, a head-to-head comparison of oral microbiome changes at a metagenomic level with longitudinal sampling has yet to be performed in these patient cohorts. Methods and Materials: To comparatively analyze oral microbiome shifts during head and neck radiation therapy, a prospective pilot cohort study was performed at the Maryland Proton Treatment Center and the University of Maryland Marlene and Stewart Greenebaum Comprehensive Cancer Center. A longitudinal metagenomic comparative analysis of oral microbiome shifts was performed at three time points (pre-radiation, during radiation, and immediately post-radiation). Head and neck cancer patients receiving proton radiation (n = 4) were compared to photon radiation (n = 4). Additional control groups included healthy age- and sex-matched controls (n = 5), head and neck cancer patients who never received radiation therapy (n = 8), and patients with oral inflammatory disease (n = 3). Results: Photon therapy patients presented with lower microbial alpha diversity at all timepoints, and there was a trend towards reduced species richness as compared with proton therapy. Healthy controls and proton patients exhibited overall higher and similar diversity. A more dysbiotic state was observed in patients receiving photon therapy as compared to proton therapy, in which oral microbial homeostasis was maintained. Mucositis was observed in 3/4 photon patients and was not observed in any proton patients during radiation therapy. The bacterial de novo pyrimidine biosynthesis pathway and the nitrate reduction V pathway were comparatively higher following photon exposure. These functional changes in bacterial metabolism may suggest that photon exposure produces a more permissive environment for the proliferation of pathogenic bacteria. Conclusion: Oral microbiome dysbiosis in patients receiving photon radiation may be associated with increased mucositis occurrence. Proton radiation therapy for head and neck cancer demonstrates a safer side effect profile in terms of oral complications, oral microbiome dysbiosis, and functional metabolic status.

The social context of burning mouth syndrome: an exploratory pilot study of stigma, discrimination, and pain.

BACKGROUND: The social context of burning mouth syndrome (BMS) has received little attention in the scientific literature. However, social psychological theory and insights from those with lived experiences suggest that people living with BMS experience compounding effects of stigma related to their pain, diagnosis (or lack thereof), and intersectional identities. OBJECTIVE: Our aim is to provide initial evidence and to motivate new directions for research on BMS. Here, we present the results of an exploratory pilot study (n = 16) of women living with BMS in the United States. METHODS: Participants completed self-report measures of stigma, discrimination, and pain, as well as laboratory assessments of pain through quantitative sensory testing. RESULTS: Results indicate a high prevalence of internalized BMS stigma, experience of BMS-related discrimination from clinicians, and gender stigma consciousness in this population. Moreover, results provide initial evidence that these experiences are related to pain outcomes. The most robust pattern of findings is that internalized BMS stigma was related to greater clinical pain severity, interference, intensity, and unpleasantness. CONCLUSION: Given the prevalence and pain-relevance of intersectional stigma and discrimination identified in this pilot study, lived experience and social context should be incorporated into future research on BMS.

Long-Term Post-COVID-19 Associated Oral Inflammatory Sequelae.

The oral cavity remains an underappreciated site for SARS-CoV-2 infection despite the myriad oral conditions observed in COVID-19 patients. Recently, replicating SARS-CoV-2 was found inside salivary epithelial cells resulting in inflammation and atrophy of salivary glands. Saliva possesses healing properties crucial for maintaining the health of the oral mucosa. Specifically, salivary antimicrobial peptides, most notable, histatin-5 exclusively produced in salivary glands, plays a vital role in innate immunity against colonizing microbial species. The demonstration of SARS-CoV-2 destruction of gland tissue where histatin-5 is produced strongly indicate that histatin-5 production is compromised due to COVID-19. Here we present a case of a patient presenting with unexplained chronic oral dysesthesia and dysgeusia post-recovery from COVID-19. To explore potential physiological mechanisms behind the symptoms, we comparatively analyzed saliva samples from the patient and matched healthy subject for histatin-5 and key cytokines. Findings demonstrated significantly reduced histatin-5 levels in patient's saliva and activation of the Th17 inflammatory pathway. As histatin-5 exhibits potent activity against the opportunistic oral pathogen Candida albicans, we evaluated saliva potency against C. albicans ex vivo. Compared to control, patient saliva exhibited significantly reduced anti-candidal efficacy. Although speculative, based on history and salivary analysis we hypothesize that salivary histatin-5 production may be compromised due to SARS-CoV-2 mediated salivary gland destruction. With the current lack of emphasis on implications of COVID-19 on oral health, this report may provide lacking mechanistic insights that may lead to reassessment of risks for oral opportunistic infections and mucosal inflammatory processes in acutely-ill and recovered COVID-19 patients.

Time of Day Influences Psychophysical Measures in Women With Burning Mouth Syndrome.

Burning mouth syndrome (BMS) is a chronic orofacial pain condition that mainly affects postmenopausal women. BMS type I patients report little to no spontaneous pain in the morning and increases in pain through the day, peaking in the afternoon. Quantitative sensory testing (QST) findings from BMS type 1 patients are inconsistent as they fail to capture this temporal variation. We examined how QST in BMS type 1 (n = 18) compared to healthy participants (n = 33) was affected by time of day. QST of the face and forearm included warmth detection threshold (WDT), cold detection threshold (CDT), and heat pain thresholds (HPT), ratings of suprathreshold heat, and pressure pain thresholds (PPT), and was performed twice: once in the morning and once in the afternoon. Compared to healthy participants, BMS patients had higher pain sensitivity to phasic heat stimuli at most temperatures (35°C U = 126.5, p = 0.0006, 39°C U = 186.5, p = 0.0386, 41°C U = 187.5, p = 0.0412, 43°C U = 171, p = 0.0167, 45°C U = 168.5, p = 0.0146) on the forearm, but no differences in pain thresholds (HPT and PPT) regardless of time of day or body area tested. BMS patients had higher WDT (U = 123, p = 0.0172), and lower CDT (U = 98, p = 0.0021) of the forearm and lower WDT of the face (U = 55, p = 0.0494). The differences in forearm WDT (U = 71.5, p = 0.0113) and CDT (U = 70, p = 0.0096) were most pronounced in the morning. In summary, BMS type I patients had increased pain sensitivity on the forearm, but no differences in pain thresholds on the face or forearm. Patients also showed altered thermal sensitivity, which depended on body area tested (heightened in the orofacial region but blunted on the forearm), and was more pronounced in the morning plausibly due to hypervigilance.

Prospective Observational Study of Bisphosphonate-Related Osteonecrosis of the Jaw in Multiple Myeloma: Microbiota Profiling and Cytokine Expression.

PURPOSE: Define incidence and risk factors of osteonecrosis of the jaw (ONJ) and explore oral microbial signatures and host immune response as reflected by cytokine changes in saliva and serum in multiple myeloma (MM) patients on bisphosphate (BP) therapy. PATIENTS AND METHODS: A single center observational prospective study of MM patients (n = 110) on >2 years of BP, none had ONJ at enrollment. Patients were followed every 3 months for 18 months with clinical/dental examination and serial measurements of inflammatory cytokines, bone turnover markers, and angiogenic growth factors. Oral microbiota was characterized by sequencing of 16S rRNA gene from saliva. RESULTS: Over the study period 14 patients (13%) developed BRONJ, at a median of 5.7 years (95% CI: 1.9-12.0) from MM diagnosis. Chronic periodontal disease was the main clinically observed risk factor. Oral microbial profiling revealed lower bacterial richness/diversity in BRONJ. Streptococcus intermedius, S. mutans, and S. perioris were abundant in controls; S. sonstellatus and S anginosus were prevalent in BRONJ. In the saliva, at baseline patients who developed BRONJ had higher levels of MIP-1ß; TNF-α and IL-6 compared to those without BRONJ, cytokine profile consistent with M-1 macrophage activation. In the serum, patients with BRONJ have significantly lower levels of TGF beta and VEGF over the study period. CONCLUSION: Periodontal disease associated with low microbial diversity and predominance of invasive species with a proinflammatory cytokine profile leading to tissue damage and alteration of immunity seems to be the main culprit in pathogenesis of BRONJ.

Stomatitis associated with mammalian target of rapamycin inhibition: A review of pathogenesis, prevention, treatment, and clinical implications for oral practice in metastatic breast cancer.

BACKGROUND: Patients with metastatic breast cancer may develop oral morbidities that result from therapeutic interventions. Mammalian target of rapamycin (mTOR) inhibitor-associated stomatitis (mIAS) is a common adverse event (AE), secondary to mTOR inhibitor therapy, that can have a negative impact on treatment adherence, quality of life, and health care costs. A multidisciplinary team approach is important to minimize mIAS and to maximize treatment benefits to patients with breast cancer. In this review, we discuss the pathophysiology, diagnosis, and natural history of mIAS. Current and new management strategies for the prevention and treatment of mIAS are described in the context of fostering a coordinated team care approach to optimizing patient care. TYPES OF STUDIES REVIEWED: The authors conducted a PubMed search from 2007 through 2017 using the terms "stomatitis," "mIAS," "everolimus," "mTOR," "metastatic breast cancer," and "oral care." They selected articles published in peer-reviewed journals that reported controlled trials and evidence-based guidelines. RESULTS: mIAS can be distinguished from mucositis caused by cytotoxic chemotherapy or radiotherapy on the basis of cause, clinical presentation, and treatment paradigms. Specific preventive and therapeutic management strategies can be implemented across the continuum of patient oral health care. PRACTICAL IMPLICATIONS: Oral health care providers are on the frontline of oral health care for patients with metastatic breast cancer and are uniquely positioned to provide patient education, advocate accurate reporting of mIAS, and support early identification, monitoring, and prompt intervention to mitigate the severity and duration of this manageable, potentially dose-limiting AE.

Prevention of everolimus-related stomatitis in women with hormone receptor-positive, HER2-negative metastatic breast cancer using dexamethasone mouthwash (SWISH): a single-arm, phase 2 trial.

BACKGROUND: Stomatitis is a class effect associated with the inhibition of mTOR and is associated with everolimus therapy for breast cancer. Topical steroids might reduce stomatitis incidence and severity, and the need for dose reductions and interruptions of everolimus. Anecdotal use of topical steroid oral prophylaxis has been reported in patients with breast cancer. We aimed to assess dexamethasone-based mouthwash for prevention of stomatitis in patients with breast cancer. METHODS: This US-based, multicentre, single-arm, phase 2 prevention study enrolled women aged 18 years and older with postmenopausal status who had histologically or cytologically confirmed metastatic hormone receptor-positive, HER2-negative breast cancer. Beginning on day 1 of cycle 1, patients received everolimus 10 mg plus exemestane 25 mg daily, with 10 mL of alcohol-free dexamethasone 0·5 mg per 5 mL oral solution (swish for 2 min and spit, four times daily for 8 weeks). After 8 weeks, dexamethasone mouthwash could be continued for up to eight additional weeks at the discretion of the clinician and patient. The primary endpoint was incidence of grade 2 or worse stomatitis by 8 weeks assessed in the full analysis set (patients who received at least one dose of everolimus and exemestane and at least one confirmed dose of dexamethasone mouthwash) versus historical controls from the BOLERO-2 trial (everolimus and exemestane treatment in patients with hormone receptor-positive advanced breast cancer who were not given dexamethasone mouthwash for prevention of stomatitis). This trial is registered at ClinicalTrials.gov, number NCT02069093. FINDINGS: Between May 28, 2014, and Oct 8, 2015, we enrolled 92 women; 85 were evaluable for efficacy. By 8 weeks, the incidence of grade 2 or worse stomatitis was two (2%) of 85 patients (95% CI 0·29-8·24), versus 159 (33%) of 482 patients (95% CI 28·8-37·4) for the duration of the BOLERO-2 study. Overall, 83 (90%) of 92 patients had at least one adverse event. The most frequently reported grade 3 and 4 adverse events in the safety set were hyperglycaemia (seven [8%] of 92 patients), rash (four [4%]), and dyspnoea (three [3%]). Serious adverse events were reported in 20 (22%) patients; six (7%) were deemed treatment related, with dyspnoea (three [3%]) and pneumonia (two [2%]) reported most frequently. 12 (13%) of 92 patients had adverse events suspected to be related to treatment that led to discontinuation of everolimus and exemestane (the most common were rash, hyperglycaemia, and stomatitis, which each affected two [2%] patients). INTERPRETATION: Prophylactic use of dexamethasone oral solution substantially reduced the incidence and severity of stomatitis in patients receiving everolimus and exemestane and could be a new standard of oral care for patients receiving everolimus and exemestane therapy. FUNDING: Novartis Pharmaceuticals Corporation.

Oral mucosal injury caused by mammalian target of rapamycin inhibitors: emerging perspectives on pathobiology and impact on clinical practice.

In recent years oral mucosal injury has been increasingly recognized as an important toxicity associated with mammalian target of rapamycin (mTOR) inhibitors, including in patients with breast cancer who are receiving everolimus. This review addresses the state-of-the-science regarding mTOR inhibitor-associated stomatitis (mIAS), and delineates its clinical characteristics and management. Given the clinically impactful pain associated with mIAS, this review also specifically highlights new research focusing on the study of the molecular basis of pain. The incidence of mIAS varies widely (2-78%). As reported across multiple mTOR inhibitor clinical trials, grade 3/4 toxicity occurs in up to 9% of patients. Managing mTOR-associated oral lesions with topical oral, intralesional, and/or systemic steroids can be beneficial, in contrast to the lack of evidence supporting steroid treatment of oral mucositis caused by high-dose chemotherapy or radiation. However, steroid management is not uniformly efficacious in all patients receiving mTOR inhibitors. Furthermore, technology does not presently exist to permit clinicians to predict a priori which of their patients will develop these lesions. There thus remains a strategic need to define the pathobiology of mIAS, the molecular basis of pain, and risk prediction relative to development of the clinical lesion. This knowledge could lead to novel future interventions designed to more effectively prevent mIAS and improve pain management if clinically significant mIAS lesions develop.

Development and In Vivo Evaluation of a Novel Histatin-5 Bioadhesive Hydrogel Formulation against Oral Candidiasis.

Oral candidiasis (OC), caused by the fungal pathogen Candida albicans, is the most common opportunistic infection in HIV(+) individuals and other immunocompromised populations. The dramatic increase in resistance to common antifungals has emphasized the importance of identifying unconventional therapeutic options. Antimicrobial peptides have emerged as promising candidates for therapeutic intervention due to their broad antimicrobial properties and lack of toxicity. Histatin-5 (Hst-5) specifically has exhibited potent anticandidal activity indicating its potential as an antifungal agent. To that end, the goal of this study was to design a biocompatible hydrogel delivery system for Hst-5 application. The bioadhesive hydroxypropyl methylcellulose (HPMC) hydrogel formulation was developed for topical oral application against OC. The new formulation was evaluated in vitro for gel viscosity, Hst-5 release rate from the gel, and killing potency and, more importantly, was tested in vivo in our mouse model of OC. The findings demonstrated a controlled sustained release of Hst-5 from the polymer and rapid killing ability. Based on viable C. albicans counts recovered from tongues of treated and untreated mice, three daily applications of the formulation beginning 1 day postinfection with C. albicans were effective in protection against development of OC. Interestingly, in some cases, Hst-5 was able to clear existing lesions as well as associated tissue inflammation. These findings were confirmed by histopathology analysis of tongue tissue. Coupled with the lack of toxicity as well as anti-inflammatory and wound-healing properties of Hst-5, the findings from this study support the progression and commercial feasibility of using this compound as a novel therapeutic agent.

Alternative Therapeutic Approach in the Treatment of Oral Pyogenic Granuloma.

Pyogenic granulomas (PGs) in the oral cavity present as an inflammatory hyperplasia usually caused by trauma, hormonal imbalance, chronic irritation, or as the response to a wide variety of drugs. PGs with atypical presentation and behavior may clinically mimic malignant tumors. Thus, histological examination is required to rule out cancer development. Lesions in the oral cavity have been described to be either an isolated entity or present in multiple forms and with multiple recurrences. Conservative surgical excision is the standard choice of treatment in almost every scenario. However, the severity of the lesions and the affected sites often challenge surgical treatment. In this report, we describe the clinical scenario of a recurrent PG, where surgical excision of the lesion was questioned. As an alternative, we describe a noninvasive approach with lesional steroid injections.

Recognition and Management of Oral Mucosal Injury Caused by Mammalian Target of Rapamycin Inhibitors: A Case Series.

The mammalian target of rapamycin inhibitors (mTORIs) everolimus and temsirolimus are approved by the US Food and Drug Administration (FDA) for the treatment of various forms of advanced cancer, and the mTORI sirolimus is approved as an immunosuppressive agent for the prophylaxis of organ rejection in patients receiving renal transplants. The oral lesions associated with mTORI toxicity are distinct from the well-documented chemotherapy- and radiotherapy-induced mucositis, but they may often be misdiagnosed by medical oncologists or transplant physicians, potentially resulting in inappropriate management of this complication. mTORI-associated oral mucosal injury appears to be dose related, and its onset is consistently earlier than conventional mucositis associated with chemotherapy or radiation therapy. Although the lesions appear to resolve within approximately 2 weeks and do not seem to recur as severely with subsequent courses of therapy, the reduction in a patient's quality of life as a result of oral pain that affects the intake of nutritional foods should be taken into consideration. We report three cases that illustrate the complexity involved in the early assessment, referral, and appropriate management of mTORI-associated oral mucosal injury. Corticosteroids appear to be very useful in managing and perhaps preventing these lesions, whereas this approach has never shown efficacy in conventional chemotherapy-related mucositis. Early intervention to reduce the mTORI-associated oral mucosal injury is important to diminish the need for dose alterations of mTORIs and, therefore, to improve patient outcomes.

Lactoferrin levels in gingival crevicular fluid and saliva of HIV-infected patients with chronic periodontitis.

AIM: This study compared lactoferrin (LF) levels in the gingival crevicular fluid (GCF) and saliva between HIV-infected and noninfected patients with chronic periodontitis. METHODS: For each subject, LF levels were analyzed in one shallow site (SS; PD ≤3 mm), one deep site (DS; PD >5 mm) and in resting whole saliva. Two groups, 28 HIV-infected and 10 noninfected, were selected. RESULTS: Although the salivary LF levels were higher in HIV-infected than in noninfected individuals, especially in AIDS patients, this was not statistically significant (P > 0.05). Subgingival LF levels for SS and DS were lower among HIV-infected individuals, although AIDS patients showed the lowest levels. Age, smoking, gender, T CD4 lymphocytes levels and viral load did not influence subgingival LF levels, neither for SS nor for DP. Positive fungal culture was observed in 24 HIV-infected patients, but only observed in one in the control group. Overall, LF concentration was significantly higher in DS than SS, both in HIV-infected and noninfected individuals (P < 0.05) and salivary LF levels were always higher than GCF levels. CONCLUSION: The data indicate that LF levels in the GCF and saliva are not different between HIV-infected and noninfected patients with chronic periodontitis.

Altered structure and function in the hippocampus and medial prefrontal cortex in patients with burning mouth syndrome.

Burning mouth syndrome (BMS) is a debilitating, idiopathic chronic pain condition. For many BMS patients, burning oral pain begins in late morning and becomes more intense throughout the day, peaking by late afternoon or evening. We investigated brain gray matter volume (GMV) with voxel-based morphometry (VBM), white matter fractional anisotropy (FA) with diffusion tensor imaging (DTI), and functional connectivity in resting state functional MRI (rsfMRI) in a tightly screened, homogeneous sample of 9 female, postmenopausal/perimenopausal BMS patients and 9 matched healthy control subjects. Patients underwent 2 scanning sessions in the same day: in the morning, when ongoing pain/burning was low, and in the afternoon, when pain/burning was significantly higher. Patients had increased GMV and lower FA in the hippocampus (Hc), and decreased GMV in the medial prefrontal cortex (mPFC). rsfMRI revealed altered connectivity patterns in different states of pain/burning, with increased connectivity between mPFC (a node in the default mode network) and anterior cingulate cortex, occipital cortex, ventromedial PFC, and bilateral Hc/amygdala in the afternoon compared with the morning session. Furthermore, mPFC-Hc connectivity was higher in BMS patients than control subjects for the afternoon but not the morning session. mPFC-Hc connectivity was related to Beck depression inventory scores both between groups and between burning states within patients, suggesting that depression and anxiety partially explain pain-related brain dysfunction in BMS. Overall, we provide multiple lines of evidence supporting aberrant structure and function in the mPFC and Hc, and implicate a circuit involving the mPFC and Hc in regulating mood and depressive symptoms in BMS.

Impaired Histatin-5 Levels and Salivary Antimicrobial Activity against C. albicans in HIV Infected Individuals.

HIV-infected individuals constitute a population highly susceptible to opportunistic infections, particularly oral candidiasis caused by the most pathogenic human fungal species Candida albicans. Host-produced salivary antimicrobial peptides are considered to be an important part of the host innate immune system involved in protection of the oral cavity against colonization and infection by microbial species. Histatin-5 (Hst-5) specifically has exhibited potent anti-candidal properties in vitro. However, its importance in protecting the oral mucosa against candidal colonization and importantly, its contribution to the observed enhanced susceptibility of HIV-infected individuals to candidiasis has not been previously investigated. To that end, a novel immunoassay was used to demonstrate significant decrease in salivary Hst-5 levels in HIV+ individuals concomitant with enhanced candidal prevalence. Further, saliva's anti-candidal potency was found to be proportional to Hst-5 concentration and significantly compromised in HIV+ subjects compared to controls. The key role for Hst-5 was further confirmed upon exposure to the Hst-5-specific antibody where saliva's initial killing activity was substantially compromised. Combined, these findings identify Hst-5 as a key anti-candidal salivary component and demonstrate its decreased levels in HIV infection providing new insights into oral Innate immune defense mechanisms and the enhanced susceptibility of HIV+ individuals to oral candidiasis.

A review of common oral pathology lesions, with a focus on periodontology and implantology.

The recognition, diagnosis, and management of common oral conditions requires knowledge of the lesion's clinical characteristics as well as the underlying pathology of the lesion. A thorough medical history, knowledge of normal anatomy, and a complete head and neck examination are necessary for the early recognition of oral lesions. Once any oral pathology is noted, clinical characteristics of size, location, texture, color, symptoms, and duration are necessary to arrive at a working plan and eventually a definitive diagnosis. In the end, the diagnosis often requires a biopsy or culture of the lesion. The clinical and histopathologic and/or mycologic correlation renders a final diagnosis leading to therapeutic options. A thorough knowledge of common oral lesions will allow the dentist/specialist to provide proper therapy or allow for referral to an oral medicine or oral surgery specialist. This review covers common infectious, reactive/traumatic, white, red, and bone lesions, as well as the vesiculobullous/desquamative gingival conditions, with a focus on periodontology and implantology. We cover the etiology, clinical features, histopathology, and treatment of each oral pathological condition.

Zoledronic acid directly suppresses cell proliferation and induces apoptosis in highly tumorigenic prostate and breast cancers.

BACKGROUND: Bisphosphonates (BPs) were designed for the prevention of skeletal-related events secondary to bone metastases. The purpose of this study was to show that zoledronic acid (ZA) directly eradicates highly tumorigenic and potentially metastatic cancer cells. MATERIALS AND METHODS: Human prostate and breast highly tumorigenic (PC3, MCF 7) and low- or non-tumorigenic (LNCaP, MCF 10a) cell lines, respectively, were exposed to different concentrations of ZA (0-10 µM). Reverse transcriptase double quantitative polymerase chain reaction was used for quantitative gene expression analysis. Apoptosis and cell proliferation were determined using microscopic observation and MTS assays. Western blot was used to confirm the translational effects of apoptotic genes on protein expression. RESULTS: Human prostate and breast highly tumorigenic (PC3, MCF 7) and low- or non-tumorigenic (LNCaP, MCF 10a) cell lines, respectively, showed multiple genes demonstrating differential expressions, including TRAF, TRADD, BCL2, CASPASES and IAP families. Increasing ZA concentrations showed a greater concentration-time response on cell proliferation and apoptosis in the highly tumorigenic cells. These results were confirmed by both reversing and enhancing the effect of ZA on cell proliferation with caspase 3, 7 or survivin siRNA, respectively. Pro-apoptotic proteins bax and caspase 2, 3, 7 and 9 were up-regulated, while the anti-apoptotic proteins bcl2, birc3 and survivin were down-regulated only in the highly tumorigenic cells. CONCLUSIONS: This explains the ability of ZA to inhibit bony metastasis in highly tumorigenic cells compared with the low- or non-tumorigenic cells through a significant decrease in cell proliferation and increase in apoptosis through gene-regulated and translational-mediated down-regulation of survivin coupled with the inhibition of caspase 3 or 7. This has significant implications toward understanding the pharmacophysiology of BPs in metastasis and supports the clinically observed effect of BPs when administered adjunctively with anticancer drugs such as cyclophosphamide/methotrexate/5-fluorouracil, epirubicin in combination with cyclophosphamide or docetaxel, and doxorubicin.