氯黴素引發細胞老化與癌化機轉之探討

Abstract

近年來抗生素濫用的問題不只造成醫療資源的浪費，也增加了微生物對第一、二線抗生素的抗藥性，更重要的是，抗生素的濫用不只增加肝臟代謝上的負擔，也會增加致癌的風險，然而目前並沒有直接的證據可以證明抗生素過度和不當的使用會致癌。 許多的抗生素，包括氯黴素和四環黴素等，都是透過抑制原核生物的蛋白質生合成來達到抑菌和制菌的功效，而哺乳類動物的粒線體由於具有類似原核生物的環狀遺傳物質，並且具有自體的蛋白質生合成系統，因此這一類的抗生素在使用上雖然能有效的抑制原核生物的蛋白質生合成，同時也可能影響粒線體的蛋白質生合成。粒線體的遺傳物質孕藏了13個與氧化磷酸化有關的蛋白體，因此，粒線體蛋白質生合成的損害會影響細胞內能量的產生和許多與粒線體有關的生理反應。本篇研究是以氯黴素的處理引發粒線體逆境，進而探討由粒線體逆境所引發的反應。 我們選用HepG2和H1299兩株細胞株給予氯黴素(10~100 μg/ml)的處理，相較於對照組我們發現在氯黴素處理的細胞，(1)由粒線體自行轉譯的蛋白含量明顯的下降，而由細胞核和細胞質內核糖體調控合成的粒線體蛋白質則不受影響，(2)細胞內ATP的含量也隨著氯黴素的處理而降低，(3)細胞的生長潛力也趨緩，細胞的生長週期停滯在G1時期，(4)細胞的形態呈現扁平和不規則的形狀，(5)並且對細胞老化的生物指標SAβ-Gal的活化，這樣的結果顯示，氯黴素所引發的粒線體逆境會增加細胞內鈣離子的濃度並且會導致細胞走向老化。細胞的老化會影響其基因表現的模式，我們發現與細胞老化有關的p21 (調控細胞週期)、Galectin-3 (細胞基質組成)、MMP-3和MMP-13 (胞外基質金屬蛋白酶)、RyR (鈣離子通道) 等基因的表現較對照組明顯的提高。 接著我們嘗試探討由氯黴素所誘導的p21蛋白所引發的生理意義， p21除了抑制細胞週期的進行外也具有抑制細胞凋亡的功能。我們發現氯黴素的處理可以抑制Mitomycin C所引發的caspase 3的活化、PARP的斷裂和細胞的凋亡，而當細胞轉染p21的反義寡核苷酸或siRNA時，則可以同時抑制由氯黴素所造成p21的表現和對細胞凋亡的保護作用，這樣的結果顯示 p21是造成氯黴素拮抗細胞凋亡的重要因子。我們也發現氯黴素會增加p21 mRNA的穩定性和改變p21蛋白在細胞內的分佈。這些結果均顯示，氯黴素所引發的粒線體逆境會透過p21的表現和分佈來抑制caspase 3的活化，進而阻斷致凋亡訊息的傳遞，增加細胞在逆境下的存活率。 我們也嘗試探討氯黴素是否會影響細胞外基質金屬蛋白酶 (MMP) 的表現，我們發現氯黴素會促進MMP-13的表現，同時我們也證明氯黴素會活化MAPKs (ERK, JNK, p38-MAPK) 和PI-3K，其中PI-3K的活性與JNK的活化有密切的關聯，JNK的活化會接著對其受質c-Jun蛋白進行磷酸化的修飾，磷酸化的c-Jun會轉位到細胞核中並活化轉錄因子AP-1並啟動MMP-13的表現。因此，氯黴素誘發MMP-13的表現可以被JNK、PI-3K和AP-1的抑制劑SP600125、LY294002和薑黃素所抑制，MMP-13可以有效的降解細胞外的基質，因此被認為和腫瘤的侵犯和轉移有關，我們發現氯黴素處理後的細胞其侵犯能力明顯增加，而MMP-13的抑制劑CL-82158則可以完全抑制其侵犯能力。因此我們認為氯黴素會藉由誘導MMP-13的表現和分泌，增強癌細胞的侵犯能力。 我們的實驗同時也證明上述的現象不只存在於氯黴素，在其它的抗生素，像doxycycline、minocycline 和clindamycin都有類似的情形。因此，我們提供了確切的證據證明，雖然這些抗生素不具有致突變性，但仍可以透過抑制細胞凋亡和促進侵犯能力等機轉，間接的增加細胞在癌化過程中的潛力，因此我們認為在臨床上此類抗生素的使用必須更加謹慎小心。

The overdose and abuse of antibiotics have been a worldwide problem that it not only caused the antibiotics resistance in microbes but increased the risk in cancer development. However, there is no direct evidence about the relationship between carcinogenesis and antibiotics abuse. Several antibiotics, such as tetracyclines and chloramphenicol, can inhibit both bacterial and mitochondrial protein synthesis. The mitochondria are maternally inherited organelles and possess a circular extra-nuclear genome that encoded 13 polypeptides of mitochondrial oxidative phosphorylayion complexes. Hence, the damage in mitochondrial translation may cause ATP depletion and a series responses. In present study, we used the chloramphenicol to stress on mitochondria and to investigate the chloramphenicol-induced cellular responses. We found that the chloramphenicol inhibited mitochondria-encoded protein synthesis specifically and decreased the cellular ATP level and the proliferative capacity. The chloramphenicol-treated cells also halted cell cycle at G0/G1 phase, elevated intracellular Ca2+ concentration, changed cell morphology to senescence-like shape and showed the appearance of senescence biomarker SA-β Gal. The chelation of intracellular Ca2+ will blocked the SA-β Gal activation suggested the involvement of Ca2+ in chloramphenicol-mediated senescence biogenesis. Moreover, the senescence-associated genes such as p21, galectin-3, matrix metalloproteinase-3 and -13 and the Ca2+ channel RyR were over-expressed in chloramphenicol-treated cells. Subsequently, we attempted to investigate the physiological function p21 in chloramphenicol-treated cells. Pretreatment of HepG2 and H1299 cells with chloramphenicol rendered the cells resistant to mitomycin-induced apoptosis. Both mitomycin-induced caspase 3 activity and PARP activation were also inhibited. Both p21 antisense and siRNA could restore apoptotic-associated caspase 3 activity, PARP activation, and the sensitivity to mitomycin-induced apoptosis. Cellular levels of the p21 protein and mRNA were increased through a p53-independent pathway, possibly due to the stabilization of p21 mRNA in chloramphenicol-treated cells. The p21 was redistributed from the perinuclear region to the cytoplasm and co-localized with mitochondrial marker protein. These findings suggested that mitochondrial stress causes resistance to apoptosis through a p21-dependent pathway. We also found the treatment of chloramphenicol could enhance MMP-3 and MMP-13 expression in JNK, PI-3K/Akt and AP-1 dependent manner. The chloramphenicol-mediated expression of MMP-13 could be inhibited by SP600125, LY294002 and curcumin. The JNK and c-Jun protein were phosphorylated in chloramphenicol-treated cells and the phospho-c-Jun protein will translocate into nucleus then activate MMP-13 promoter through AP-1 consensus site. The MMP-13 has been demonstrated in cancer invasion and metastasis. Chloramphenicol-treated cells showed strong invasive potential than untreated cells and the increase in invasive capacity could be completely prevented by MMP-13 inhibitor CL-82158. These findings suggested that chloramphenicol administration may accelerate cancer cell invasion through a MMP-13 dependent mechanism. The similar effects also observed in minocycline, doxycycline and clindamycin. Our studies provided strong evidence for the antibiotics abuse in cancer development. The apoptotic resistance and MMP-13 dependent cell invasion may enhance cancer cell survival and accelerate cancer cell invasion, respectively. Finally, the clinical significant of present studies caution the clinical use of these antibiotics should be more carefully, especially during cancer chemotherapy.