Faculty Bibliography

Persistent paraprotein production in plasma cells necessitates a highly developed rough endoplasmic reticulum (ER) that is unusually susceptible to perturbations in protein synthesis. This biology is believed to account for the exquisite sensitivity of multiple myeloma (MM) to the proteasomal inhibitor bortezomib (BTZ). Despite remarkable response rates to BTZ in MM, BTZ carries the potential for serious side-effects and development of resistance. We, therefore, sought to identify therapeutic combinations that effectively disrupt proteostasis in order to provide new potential treatments for MM. We found that sulforaphane, a dietary isothiocyanate found in cruciferous vegetables, inhibits TNFalpha-induced Ikappabeta proteasomal degradation in a manner similar to BTZ. Like BTZ, sulforaphane synergistically enhances the cytotoxicity of arsenic trioxide (ATO), an agent with clinical activity in MM. ATO and sulforaphane co-treatment augmented apoptotic induction as demonstrated by cleavage of caspase-3, -4 and PARP. The enhanced apoptotic response was dependent upon production of reactive oxygen species (ROS) as demonstrated by glutathione depletion and partial inhibition of the apoptotic cascade after pretreatment with the radical scavenger N-acetyl-cysteine (NAC). Combination treatment resulted in enhanced ER stress signaling and activation of the unfolded protein response (UPR), indicative of perturbation of proteostasis. Specifically, combination treatment caused elevated expression of the molecular chaperone HSP90 (heat shock protein 90) along with increased PERK (protein kinase RNA-like endoplasmic reticulum kinase) and eIF2alpha phosphorylation and XBP1 (X-box binding protein 1) splicing, key indicators of UPR activation. Moreover, increased splicing of XBP1 was apparent upon combination treatment compared to treatment with either agent alone. Sulforaphane in combination with ATO effectively disrupts protein homeostasis through ROS generation and induction of ER stress to culminate in inhibition of protein secretion and apoptotic induction in MM. Our results suggest that sulforaphane deserves further investigation in combination with ATO in the treatment of MM.

Vitiligo is characterized by depigmented skin patches caused by loss of epidermal melanocytes. Oxidative stress may have a role in vitiligo onset, while autoimmunity contributes to disease progression. In this study, we sought to identify mechanisms that link disease triggers and spreading of lesions. A hallmark of melanocytes at the periphery of vitiligo lesions is dilation of the endoplasmic reticulum (ER). We hypothesized that oxidative stress results in redox disruptions that extend to the ER, causing accumulation of misfolded peptides, which activates the unfolded protein response (UPR). We used 4-tertiary butyl phenol and monobenzyl ether of hydroquinone, known triggers of vitiligo. We show that expression of key UPR components, including the transcription factor X-box-binding protein 1 (XBP1), is increased following exposure of melanocytes to phenols. XBP1 activation increases production of immune mediators IL6 and IL8. Co-treatment with XBP1 inhibitors reduced IL6 and IL8 production induced by phenols, while overexpression of XBP1 alone increased their expression. Thus, melanocytes themselves produce cytokines associated with activation of an immune response following exposure to chemical triggers of vitiligo. These results expand our understanding of the mechanisms underlying melanocyte loss in vitiligo and pathways linking environmental stressors and autoimmunity.Journal of Investigative Dermatology advance online publication, 14 June 2012; doi:10.1038/jid.2012.181.

OBJECTIVE To investigate the frequency, epidemiology, clinical features, and prognostic significance of inflamed molluscum contagiosum (MC) lesions, molluscum dermatitis, reactive papular eruptions resembling Gianotti-Crosti syndrome, and atopic dermatitis in patients with MC. DESIGN Retrospective medical chart review. SETTING University-based pediatric dermatology practice. PATIENTS A total of 696 patients (mean age, 5.5 years) with molluscum. MAIN OUTCOME MEASURES Frequencies, characteristics, and associated features of inflammatory reactions to MC in patients with and without atopic dermatitis. RESULTS Molluscum dermatitis, inflamed MC lesions, and Gianotti-Crosti syndrome-like reactions (GCLRs) occurred in 270 (38.8%), 155 (22.3%), and 34 (4.9%) of the patients, respectively. A total of 259 patients (37.2%) had a history of atopic dermatitis. Individuals with atopic dermatitis had higher numbers of MC lesions (P < .001) and an increased likelihood of molluscum dermatitis (50.6% vs 31.8%; P < .001). In patients with molluscum dermatitis, numbers of MC lesions increased during the next 3 months in 23.4% of those treated with a topical corticosteroid and 33.3% of those not treated with a topical corticosteroid, compared with 16.8% of patients without dermatitis. Patients with inflamed MC lesions were less likely to have an increased number of MC lesions over the next 3 months than patients without inflamed MC lesions or dermatitis (5.2% vs 18.4%; P < .03). The GCLRs were associated with inflamed MC lesion (P < .001), favored the elbows and knees, tended to be pruritic, and often heralded resolution of MC. Two patients developed unilateral laterothoracic exanthem-like eruptions. CONCLUSIONS Inflammatory reactions to MC, including the previously underrecognized GCLR, are common. Treatment of molluscum dermatitis can reduce spread of MC via autoinoculation from scratching, whereas inflamed MC lesions and GCLRs reflect cell-mediated immune responses that may lead to viral clearance.

The pathobiology of vitiligo, characterized by the spread of depigmented skin patches due to localized melanocyte loss, is not fully understood. Oxidative stress is thought to play a role in disease onset with a subsequent autoimmune response underlying progression. We therefore sought to identify mechanisms that linked oxidative stress and autoimmune responses. Melanocytes at the periphery of vitiligo lesions have distended endoplasmic reticuli (ER). We hypothesized that oxidative stress disrupted homeostasis of the ER where oxidation/reduction reactions facilitate disulfide bond formation. As a result, misfolded peptides would accumulate, dilating the ER and activating the unfolded protein response (UPR). The UPR is a stress response pathway, initiated by three regulators (IRE1, PERK and ATF6). It first promotes cell survival, however sustained activation induces apoptosis. In order to identify a potential role for the UPR in vitiligo we dosed melanocytes with 4-tertiary butyl phenol (4- TBP) and monobenzyl ether of hydroquinone (MBEH), phenols known to trigger vitiligo. The phenols caused an increase in expression of IRE1 and PERK. PERK activation leads to enhancement of the antioxidant response by recruitment of the transcription factor NRF2 to the nucleus and increased expression of the antioxidant HMOX1. The IRE1 effector, X-box binding protein-1 (XBP1) was also activated by phenol treatment which led to increased production of interleukin-6 (IL6) and IL8, cytokines expressed at increased levels in perilesional skin in vitiligo. Treatment with XBP1 inhibitors reduced phenol-induced IL6 and IL8 production, while over-expression of active XBP1 increased their expression. Thus, chemicals known to cause vitiligo trigger a UPR-mediated increase in cytokine production. There are a number of potential roles for the UPR in vitiligo. The UPR may (i) induce apoptosis following sustained oxidative stress causing release of melanocyte specific antigens, (ii) be dysregulated resulting in a muted PERK-!

Although the precise mechanisms that trigger vitiligo remain elusive, autoimmune responses mediate its progression. The development of therapies has been impeded by a paucity of animal models, since mice lack interfollicular melanocytes, the primary targets in vitiligo. In this issue, Harris et al. describe a mouse model in which interfollicular melanocytes are retained by Kit ligand overexpression and an immune response is initiated by transplanting melanocyte-targeting CD8+ T cells.

Persistent paraprotein production in plasma cells necessitates a highly developed rough endoplasmic recticulum (ER) that is exquisitely sensitive to perturbations in protein synthesis. Targeting ER stress-related signaling has been clinically validated in the treatment of multiple myeloma (MM) as evidenced by the response to treatment with bortezomib (BTZ). Despite impressive response rates, BTZ carries the potential for serious side effects, and the development of resistance to BTZ is a clinical issue. We therefore sought to identify novel drug combinations that effectively generate ER stress. Here, we report that sulforaphane, a naturally occurring isothiocyanate found in cruciferous vegetables, synergistically enhances the cytotoxicity of arsenic trioxide (ATO), an agent that has shown clinical activity in MM, in a panel of MM cell lines. As single agents, both 1 muM sulforaphane and 0.5 muM ATO have a modest effect on cellular proliferation in a panel of MM lines. However, when the agents are administered in combination, cellular proliferation is dramatically reduced. For example, in PCNY-1 MM cells, 1 muM sulforaphane has no effect and 0.5muM ATO causes a 29% reduction in proliferation. However, when administered together, the agents enhance growth inhibition to 73%, with a CI of 0.632 indicative of synergy. Four out of 5 MM cell lines tested displayed sulforaphane and ATO synergy. Combination treatment resulted in enhanced apoptotic induction as demonstrated by cleavage of PARP. Enhanced induction of ER stress signaling and activation of the unfolded protein response (UPR) upon combination treatment was demonstrated by enhanced expression of the molecular chaperone HSP90 along with increased phosphorylation of PERK (an ER transmembrane kinase and proximal effector of the UPR) and eIF2 (translational initiation factor). Additionally, increased splicing of XBP1 (a transcription factor of UPR target genes) was apparent upon combination treatment as compared to treatment with either agent alone. Our results show that sulforaphane can synergistically sensitize MM cells to the cytotoxic effects of ATO through promotion of ER stress generating mechanisms. Based upon these promising results, further evaluation of this safe, natural product as an ATO sensitizer in a clinical trial of MM patients is warranted. Additionally, this approach holds the promise as a means to identify and clinically validate natural products effective in the treatment of MM and/or inhibition of progression of asymptomatic MM

ABSTRACT: BACKGROUND: Brain metastases afflict approximately half of patients with metastatic melanoma (MM) and small cell lung cancer (SCLC) and represent the direct cause of death in 60 to 70% of those affected. Standard of care remains ineffective in both types of cancer with the challenge of overcoming the blood brain barrier (BBB) exacerbating the clinical problem. Our purpose is to determine and characterize the potential of albendazole (ABZ) as a cytotoxic and radiosensitizing agent against MM and SCLC cells. METHODS: Here, ABZ's mechanism of action as a DNA damaging and microtubule disrupting agent is assessed through analysis of histone H2AX phosphorylation and cell cyle progression. The cytotoxicity of ABZ alone and in combination with radiation therapy is determined though clonogenic cell survival assays in a panel of MM and SCLC cell lines. We further establish ABZ's ability to act synergistically as a radio-sensitizer through combination index calculations and apoptotic measurements of poly (ADP-ribose) polymerase (PARP) cleavage. RESULTS: ABZ induces DNA damage as measured by increased H2AX phosphorylation. ABZ inhibits the growth of MM and SCLC at clinically achievable plasma concentrations. At these concentrations, ABZ arrests MM and SCLC cells in the G2/M phase of the cell cycle after 12 hours of treatment. Exploiting the notion that cells in the G2/M phase are the most sensitive to radiation therapy, we show that treatment of MM and SCLC cells treated with ABZ renders them more sensitive to radiation in a synergistic fashion. Additionally, MM and SCLC cells co-treated with ABZ and radiation exhibit increased apoptosis at 72 hours. CONCLUSIONS: Our study suggests that the orally available antihelminthic ABZ acts as a potent radiosensitizer in MM and SCLC cell lines. Further evaluation of ABZ in combination with radiation as a potential treatment for MM and SCLC brain metastases is warranted

A six-week-old girl presented with a segmental, focally atrophic, vascular patch in the diaper area, present since birth. It had undergone minimal proliferation, but had ulcerated. Evaluation to rule out LUMBAR (Lower body hemangioma/Lipoma or other cutaneous anomalies, Urogenital anomalies, Myelopathy, Bony deformities, Anorectal/Arterial anomalies, and Renal anomalies) syndrome, which included ultrasound and Doppler examination of the abdomen, spine, and pelvis, was negative. We report a unique case of an ulcerated, segmental abortive hemangioma of the anogenital area with excellent clinical response to topical timolol gel.