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.

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.

Stress induced by buildup of misfolded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR). Failure by the UPR to restore homeostasis can trigger apoptosis. We have shown chronic UPR activation in tyrosinase and Tyrp1 mutant melanocytes, where tyrosinase is retained in the ER, without concomitant loss of viability, suggesting that melanocytes adapt to ER stress. Identifying adaptive mechanisms has implications for treatment of melanocyte disorders. For example, UPR activation may permit adaptation to hypoxia and play a role in melanomagenesis. Thus melanoma therapies targeting the UPR are being tested. The UPR may also play a role in vitiligo. Xbp1 (a key UPR transcription factor) polymorphisms are associated with increased risk of vitiligo, while ER dilation in perilesional melanocytes suggests ER stress. The UPR consists of three pathways regulated by IRE1, ATF6 and PERK. Prolonged IRE1 signaling in stressed cells promotes survival, while sustained PERK activity promotes apoptosis. OCA2 mutations result in hypopigmentation due, in part, to ER retention of tyrosinase, but do not result in loss of viability. We therefore investigated the UPR in Oca2-(null) melanocytes. Wildtype and Oca2-melanocytes were treated with ER stressors thapsigargin or tunicamycin and UPR activation was monitored by RT-PCR and Western blot analysis. Ire1 expression was increased in Oca2-melanocytes compared to wildtype cells. However, downstream signaling was not activated, with no splicing of Ire1 targeted Xbp1. Expression of Perk and its downstream effector Atf4 were decreased in Oca2-melanocytes. Upon ER stress, Ire1 expression and Xbp1 splicing increased in both cell lines indicating UPR activation. Typically, UPR activation leads to Perk-mediated phosphorylation of eIF2alpha. Remarkably, levels of p-eIF2alpha were markedly diminished in stressed Oca2-cells. Expression of proapoptotic CHOP was still induced in Oca2-melanocytes, but did not result in significant cell death. Our da!

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

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.

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