Heat-clearing herbs (HCHs) have been widely used as single drug or adjuvant therapy in cancer treatment. However, it is unclear whether HCHs induce hormetic effect in cancer cells or not. Thus, the aim of this study is to examine hormetic effect of aqueous extracts of HCH and their antagonistic effect on anticancer activity of chemotherapeutic agents. In the first part, we found that the aqueous extracts of HCH (Cortex phellodendri, Hedyotis diffusa, Houttuyniae herba, Polygoni cuspidati, Paeoniae radix, Prunella vulgaris, Scutellaria baicalensis, Sophorae flavescentis and Taraxacum mongolicum) remarkably induced hormesis in B16-F10 murine melanoma cells. Low concentrations of aqueous extracts of P. vulgaris (APV) (0.32 and 0.63 mg/mL) and S. baicalensis (ASB) (0.32 and 0.63 mg/mL) increased cell viability and attenuated apoptosis induced by camptothecin (CPT) or cis-platinum (DDP), while high concentrations of APV (5 mg/mL) and ASB (2.5 mg/mL) shown significant anticancer effect alone or in combination with chemotherapeutic agents. Treatment with APV and ASB could promote the production of reactive oxygen species (ROS) in B16-F10 cells. In addition, low concentrations of APV and ASB upregulated the PI3K/Akt/FOXO3a pathway and increased the expression of antioxidase (CAT, GSH-Px and SOD2) as well as the activities of antioxidase (CAT, GPX1 and T-SOD) in B16-F10 cells. However, high concentrations of APV and ASB showed adverse effects. The PI3K inhibitor, LYP294002, attenuated the increasing effects of APV and ASB. In the second part, we found baicalein, an active component of S. baicalensis, induced hormetic effect in B16-F10 cells, MDA-MB-231 human breast cancer cells and A549 non-small cell lung cancer cells. Low concentrations of baicalein (0.4, 0.8 and 1.6 μM) antagonized anticancer effect of CPT and DDP in B16-F10 cells and activated the PI3K/Akt and MAPK pathways, while 25 μM baicalein shown anticancer effect alone or in combination with chemotherapeutic agents. Treatment with inhibitors of ERK1/2 and PI3K pathways attenuated the hormetic effect induced by baicalin and restored the anticancer activities of CPT and DDP, suggesting that ERK1/2 and PI3K/Akt pathways played important roles in the hormetic effect of baicalein. In summary, our results suggested that HCH extracts could induce hormetic effect and antagonize the antitumor activity of chemotherapeutic agents, CPT and DDP, in iv B16-F10 cells. Therefore, we need to assess the risk of HCHs before practicing in clinical trials.

清熱藥以單藥或者輔助治療的形式已經被廣泛應用於腫瘤治療。然而清熱 藥是否能夠在腫瘤細胞中誘導低劑量毒物興奮效應尚不清楚。本研究的目的是 探究清熱藥提取物的低劑量毒物興奮效應及其拮抗化療藥的抗腫瘤活性。 在本論文的第一部分中，我們研究結果表明，9種清熱藥水提物（黃柏，白 花蛇舌草，魚腥草，虎杖，赤芍，夏枯草，黃芩，苦參，蒲公英）能在 B16- F10 小鼠黑色素瘤細胞上誘導低劑量毒物興奮效應。低劑量的夏枯草水提物 (0.32 和 0.63 mg/mL)和黃芩水提物(0.32 和 0.63 mg/mL)增加 B16-F10 細胞活力 並且拮抗化療藥喜樹堿和順鉑誘導的細胞毒性和凋亡，然而高濃度的夏枯草水 提物(5 mg/mL)和黃芩水提物(2.5 mg/mL)在單用或者聯合用藥時表現出抗腫瘤 作用。夏枯草水提物和黃芩水提物在 B16-F10 細胞上促進活性氧的產生。低濃 度的夏枯草和黃芩水提物能通過啟動 PI3K/Akt/FOXO3a 通路上調抗氧化酶（過 氧化氫酶，谷胱甘肽還原酶和超氧化物歧化酶 2）的表達和抗氧化酶（過氧化 氫還原酶，谷胱甘肽還原酶 1 和總超氧化歧化酶）的活性。而 PI3K 抑制劑 LY294002 能減弱夏枯草水提物和黃芩水提物對 B16-F10 細胞的生長促進作用。 在第二部分中，我們發現黃芩素（黃芩的主要活性成分）在 B16-F10 細胞， MDA-MB-231 人乳腺癌細胞和 A549 人非小細胞肺癌細胞上誘導了低劑量毒物 興奮效應。低濃度的黃芩素(0.4，0.8和 1.6 μM)在 B16-F10細胞上拮抗化療藥喜 樹堿和順鉑的抗腫瘤作用，並且啟動 PI3K/Akt 和 MAPK 通路。然而 25 μM 黃 芩素在單用或者聯合用藥時表現出抗腫瘤作用。ERK1/2和 MAPK通路抑制劑不 僅減弱黃芩素誘導的低劑量毒物興奮效應，而且恢復喜樹堿和順鉑的抗腫瘤活 性，說明 ERK1/2 和 PI3K/Akt 通路對黃芩素的低劑量毒物興奮效應起著重要作 用。 總之，我們的研究結果表明清熱藥提取物能在 B16-F10 細胞上誘導低劑量 毒物興奮效應並拮抗化療藥的抗腫瘤活性。