Identifying consistent intracellular biomarkers at which a potential chemopreventive may act is essential prior to initiating clinical trials. As curcumin acts on many different biomolecular targets in a variety of different cell types it is important to determine if curcumin directly affects either a few major downstream biomarkers or a multiplicity of downstream targets which may serve to explain curcumin's varying effects in different cell types. Aberrant signaling through the epidermal growth factor receptor (EGFR) plays a major role in cutaneous skin cancer progression. EGFR inhibitors have been used for SCC therapy to downregulate aberrant EGFR signaling with little change in overall survival [20
], possibly due to compensating mutations downstream of EGFR. One of these signaling pathways is PI3
K/AKT that plays a role in skin carcinogenesis and in chemotherapy resistance [21
Activated Ras/Raf signaling has also been implicated in a small percentage of SCC [22
] and can lead to activation of the MAPK pathway. Ras/Raf gain-of-function can occur through activation of ERK1 and ERK2, which are constitutively active in 70% of malignant melanoma due to RAS or BRAF activating mutations [2
]. Activated ERK1/2 is rarely seen in normal skin specimens but is shown in all cases of SCC with a positive association with the degree of malignancy and proliferative activity of SCC [23
]. In this study, Zhang et al. looked at 10 well-differentiated and 10 poorly differentiated skin SCC cases. Another study looked at activated ERK in 101 human head and neck squamous carcinoma specimens [24
]. Therefore, inhibiting ERK may be a promising approach in targeted cutaneous skin SCC therapy. Having previously determined curcumin's growth inhibitory effects in skin SCC [11
], we sought to determine whether these effects were similar to our observations in upper aerodigestive head and neck SCC (HNSCC) where curcumin inhibited the AKT/MTOR pathway through rapid curcumin-dependent inhibition of MTOR's downstream target pS6 and 4EBP1 phoshorylation [18
In this study we found significant and complete inhibition of SRB12-p9 cell proliferation after treatment with curcumin at a dose 20μ
M or higher () suggesting a highly potent anticarcinogenic effect of curcumin in skin cancer. Additionally, we found that the inhibitory effect of curcumin on skin cancer proliferation was associated with inhibition of AKT/mTOR and ERK signaling ().
In our in vivo
study, curcumin paste was formulated to penetrate human skin epidermis and dermis. However, given the thin nature of mouse skin, topical curcumin penetration was much greater such that curcumin possibly did not remain in the epidermis for a prolonged period, leading to prolonged contact with the cancer cells. The irritant nature of the cream caused the skin overlying the tumor to thicken, although this was not statistically significantly different from control (P
= 0.73). The SRB12-p9 cell line is invasive in this model [25
], producing inaccurate tumor caliper measurements due to the inability to account for the portion of the tumor that invaded into the abdominal wall. Therefore, the ex vivo
tumor weight provided a more accurate tumor size endpoint. In human skin, SCC emerges directly from the epidermal layer, unlike in our xenograft model, where tumor is encapsulated under the epidermis. We therefore anticipate a more pronounced tumor-suppressive effect of topical curcumin in humans.
The SRB12-p9 xenograft cells were more sensitive to curcumin-induced cell death and apoptosis than the surrounding normal mouse skin and grew at a much slower rate in the presence of curcumin, whether topical or systemic, compared to control. IL6 plays a central role in regulating the inflammatory response [25
]. Because IL-6 may contribute to angiogenesis and metastasis [26
], inhibition of IL-6 with topical curcumin suggests a mechanism of chemoprevention. Although curcumin has previously been shown to inhibit IL-6 in HNSCC cell lines [27
], this is the first skin cancer model investigating curcumin's inhibition of systemic IL-6. The present study demonstrates that topical curcumin reduces skin SCC tumor growth, and this effect might be explained, by the inhibition of IL-6.
In this study we demonstrated significant inhibition of several biomarkers of the AKT/mTOR pathway as well as STAT3 and ERK1/2 in SRB12-p9 cells after treatment with 20μ
M of curcumin. In our in vivo
experimentation, we observed inhibition of pERK in the curcumin-treated tumors and inhibition of pSTAT3 in the combined curcumin group. However, tumor heterogeneity and degree of dysplasia can often confound immunohistochemistry results, depending on where in the lesion the biopsy was taken. Therefore, it is important to develop serum biomarkers that can be obtained with a simple blood draw. As curcumin is a well-known anti-inflammatory agent, we measured its effects on pooled serum of treated mice and noted a decrease in IL-6 in the topical group compared to the control group. We observed that systemic curcumin did not cause a decrease in serum IL-6 levels. However, only three mice in each group were analyzed, and it is possible that statistically significant differences in IL6 levels could be detected upon analysis of greater numbers of mice in the topical and combined curcumin-treated groups compared to control mice.
As curcumin slowed progression of aggressive skin SCC xenografts and inhibited pERK expression, the ERK pathway may prove to be a key biomarker in developing topical pharmaceutical agents that prevent skin SCC tumor growth or recurrence. We observed that the overall reduction in pERK staining in the curcumin-treated tumors was not cell autonomous but rather manifested as an expansion in areas of very low or no expression, such that focal regions of intense staining remained. Alternatively, control tumors had smaller regions of low staining and a higher number of intensely staining areas. This indicates that a global reduction of pERK staining was achieved with curcumin treatment, rather than a complete shutdown. [23
] confirmed that phosphorylated ERK is overexpressed in patient skin SCC in a Caucasian population, which further supports our findings and suggests that pERK may be a useful chemoprevention biomarker.
Chronic inflammation is linked to both cancer and angiogenesis. The anti-inflammatory properties of curcumin may contribute to its potential as an effective chemopreventive agent. However, curcumin's systemic anti-inflammatory effects (reduced serum IL-6 levels) were more pronounced in topical curcumin group compared to gavage. Given these findings, it was unexpected that tumor growth was inhibited more effectively in the gavage group than in the topical group. However, there was no statistically significant difference in tumor volume between the two treatment groups. Despite this data, we speculate that local anti-inflammatory activity of topically applied curcumin contributes significantly to its chemopreventive activity, circumventing its poor systemic bioavailability.
As curcumin continues to be explored as a chemopreventive and therapeutic agent for skin cancer treatment, establishing defined biomarkers upon which curcumin acts to inhibit tumorigenesis is essential. The ERK pathway is an important protein kinase signaling cascade involved in cellular proliferation and is activated in carcinogenesis. In this study, activated pERK expression significantly increased in SCC compared to the less aggressive BCC and AK. As curcumin has been shown to inhibit activated ERKs in carcinogenesis, the present data suggests that components of the ERK pathway may prove to be key biomarkers for curcumin chemopreventive efficacy in cutaneous SCC.