The anti-cancer efficacy of sulforaphane, a natural compound derived from broccoli/broccoli sprouts, has been evaluated in various cancers. For instance, oral or intraperitoneal administration of sulforaphane inhibited the tumor growth in prostate PC-3 and pancreatic Panc-1 xenografts (33
). The risk of premenopausal breast cancer was shown to be inversely associated with broccoli consumption (35
). The orally administered sulforaphane reached mammary gland and increased the detoxification enzyme activity (36
). Additionally, it has been suggested that sulforaphane may have the potential to act against tumor resistance and relapse/recurrence (37
). A very recent study demonstrated the effectiveness of sulforaphane in abrogating pancreatic tumor resistance to TRAIL by interfering with NF-κB induced anti-apoptotic signaling (18
). Another study indicated that sulforaphane could overcome doxorubicin resistance and restore apoptosis induction in cells (38
). These findings provide a strong rationale for investigating the chemoprevention property of sulforaphane or broccoli/broccoli sprouts in clinical trials.
Increasing evidence supports the cancer stem cell theory, which states that a variety of cancers are driven and sustained by a small proportion of CSCs (8
). The concept of CSCs has profound clinical implications for cancer therapeutics and prevention (8
). Recent studies indicate that CSCs have the capacity to drive tumor resistance and relapse/recurrence (40
). Lack of efficacy of current chemotherapies in advance and metastatic disease requires novel approaches to specifically target CSC population (8
). Thus, therapies that are directed against both differentiated cancer cells and CSCs may provide advantages to treat these diseases. Researchers have found that several dietary compounds are promising chemoprevention agents against CSCs, such as curcumin (13
). Therefore, based on the chemopreventive activity of sulforaphane and the implications of CSC theory, we have utilized both in vitro
and in vivo
systems to determine whether sulforaphane acts against breast CSCs.
Several techniques have been developed to isolate and characterize breast CSCs in vitro
. Mammosphere culture was first used to isolate and expand mammary stem/progenitor cells by Dontu et al.
), based on the ability of stem/progenitor cells to grow in serum-free suspension, while differentiated cells fail to survive under the same condition (21
). By employing this technique, we have demonstrated that sulforaphane (0.5~5 μM) significantly suppressed the mammospheres formation of both SUM159 and MCF7 cells (). Another technique is to utilize cell makers, e.g., CD44+
and ALDH-positive (21
), to distinguish mammary stem/progenitor cells from differentiated cancer cells. It has been reported that as few as 500 ALDH-positive cells were able to generate a breast tumor within 40 days, while 50,000 ALDH-negative cells failed to form tumor (23
). ALDH-positive and CD44+
were identified a small overlap that has the highest tumorigenic capacity, generating tumors from as few as 20 cells (23
). In contrast, ALDH-positive cells without the CD44+
marker were able to produce tumors from 1,500 cells, whereas 50,000 CD44+
ALDH-negative cells did not (23
). Thus, we utilized Aldefluor assay to evaluate the ability of sulforaphane to target breast cancer stem/progenitor cells. We have demonstrated that sulforaphane (1~5 μM) could inhibit the tumor-initiating ALDH-positive cells in vitro
by 65% to 80% (). Of special note, concentrations of sulforaphane which inhibit stem/progenitor cells in both mammosphere formation assay and Aldefluor assay had only minimal effects on the bulk population of breast cancer cell lines, which implies the preferential targeting of stem/progenitor cells by sulforaphane.
The injection of human breast cancer cells into the mammary fat pad of immune-deficient NOD/SCID mice provides a reliable and sensitive in vivo
system for studying human breast cancer (25
). We demonstrated that sulforaphane was able to target breast CSCs in vivo
by using this xenograft model. Daily injection of sulforaphane for two weeks suppressed tumor growth in primary NOD/SCID mice and reduced ALDH-positive cell population of the tumors by ~50% (). More importantly, we found that the tumor cells derived from sulforaphane-treated mice were not able to form secondary tumors in recipient mice up to 33 days (). There are two possible reasons that may explain the difference between the 50% reduction of ALDH-positive population and the failure of tumor growth in secondary mice. One is that although ALDH-positive cells are enriched with stem/progenitor cells, not all ALDH-positive cells have tumor-initiating capacity. Another possible reason is the experimental setting we used for the primary NOD/SCID mice. We inoculated 2,000,000 SUM159 cells into the primary NOD/SCID mice, and treated them with the drug after two weeks of cell inoculation, both of which could lead to an under-estimation of the effect of sulforaphane on ALDH-positive cell population. However, the ability of CSCs to self-renew and differentiate as determined by reimplantation of primary tumor cells in secondary animals is a more definitive functional assay (6
). These are consistent with the in vitro
observation that sulforaphane preferentially targeted cancer stem/progenitor cells instead of bulk cell population. The preference of sulforaphane in killing CSCs may be significant for chemoprevention.
The well-known curcumin was shown to interfere with self-renewal pathways, Wnt and Notch, in colon and pancreatic cancer cells respectively (13
). Apple-derived quercetin and green tea epigallocatechin-gallate were reported to regulate key elements of Wnt and Notch pathways in human colon cancer cells (15
). Park et al.
previously reported that β-catenin was down-regulated in HeLa and HepG2 cells (19
). In consistent with this study, we demonstrated that sulforaphane was able to down-regulate Wnt/β-catenin self-renewal pathway in breast cancer cells, and sulforaphane-induced β-catenin phosphorylation (Ser33/Ser37/Thr41) and proteasome degradation was possibly through activation of GSK3β (). Myzak et al.
reported that sulforaphane increased β-catenin activity without altering its protein level in HDAC1-transfected HEK293 cells (45
). The differences among the studies could arise from distinct cell lines and treatment conditions.
As a chemoprevention agent, sulforaphane possesses many advantages, such as high bioavailability and low toxicity (4
). Sulforaphane from broccoli extracts is efficiently and rapidly absorbed in human small intestine, and distributed throughout the body (2
). Plasma concentrations of sulforaphane equivalents peaked 0.94~2.27 μM in humans 1 hr after a single dose of 200 μmol broccoli sprout isothiocyanates (mainly sulforaphane) (47
). A recent pilot study detected an accumulation of sulforaphane in human breast tissue, with 1.45 ± 1.12 pmol/mg for the right breast and 2.00 ± 1.95 pmol/mg for the left, in eight women who consumed broccoli sprout preparation containing 200 μmol sulforaphane about 1 hr before the surgery (36
). These concentrations of sulforaphane are expected to be effective against breast CSCs, based on our in vitro
results. Although sulforaphane itself has not been evaluated in humans, broccoli sprouts were tested for toxicity in clinical trials (4
). A Phase I trial showed that broccoli sprouts caused no significant toxicity when administered orally at 8-hr intervals for 7 days as 25 μmol isothiocyanates (mainly sulforaphane) (48
). In another study, it was well tolerated in 200 adults who consumed broccoli sprout solution containing 400 μmol glucoraphanin (precursor of sulforaphane) nightly for 2 weeks (49
). Additionally, sulforaphane at concentrations below 10 μM did not show significant effect on cell cycle arrest and apoptosis induction of human non-transformed T-lymphocytes (50
In conclusion, we have demonstrated that sulforaphane was able to target breast CSCs as determined by the mammosphere formation assay, Aldefluor assay, and tumor growth upon reimplantation in secondary mice. Furthermore, our study identified the down-regulation of Wnt/β-catenin self-renewal pathway by sulforaphane as one of the possible mechanisms for its efficacy. These studies support the use of sulforaphane for breast cancer chemoprevention. These findings provide a strong rationale for preclinical and clinical evaluation of sulforaphane or broccoli/broccoli sprouts for breast cancer therapies.
Sulforaphane, the natural compound derived from broccoli/broccoli sprouts, has been proved to possess anti-cancer activity. This study demonstrates that sulforaphane inhibits breast cancer stem cells in vitro and in vivo, which provides a strong rationale for future clinical evaluation of sulforaphane or extract of broccoli/broccoli sprouts for breast cancer chemoprevention. Breast cancer is initiated from and maintained by a small population of breast cancer stem cells. Currently available chemotherapy and radiation therapy are incapable of suppressing cancer stem cell population. Aldefluor assay and mammosphere formation assay showed that sulforaphane inhibited breast cancer stem cells in vitro. NOD/SCID mouse model exhibited that sulforaphane eliminated breast cancer stem cells in vivo.