This is possible, you can still get apoptosis without these proteins being upregulated. For example, in Caspase independent apoptosis, caspases are not cleaved. In such a case you usually see a downregulation of full length PARP (116 kDA). The only way to make sure that you have apoptosis is to do an AnnexinV/PI staining.

That caspase-3 and caspase-8 are going down is perfectly fine, because caspases get activated by proteolytic cleavage that means the amount of full length caspases will go down in the course of apoptosis. I have no explanation why cleaved PARP also goes down because cleaved PARP should actually go up.

Well, there is caspase-independent apoptosis where DNA fragmentation is achieved by the factor AIF which is released from apoptotic mitochondria (those with pores).

http://clincancerres.aacrjournals.org/content/11/9/3155.full.pdf+html

This is possible , in caspase of independant apoptosis, there are others alternative models of programmed cell death have been proposed, such as autophagy, endoplasmic reticulum stress, mitotic catastrophe.... for more information Please see: http://clincancerres.aacrjournals.org/content/11/9/3155.abstract.

Hi Laishram. The intrinsic and the extrinsic apoptotic pathways work in different ways, but have caspase- 3,6,7 as the final effectors. That said, Nicole is right in saying that caspase will be turned over due to its proteolytic cleavage. As Christiana has suggested, you could do an Annexin V+PI analysis to look at apoptosis. I would also do a caspase activity assay (there are several kits available). If you performed a time course of caspase activity, it could tell you if the caspases were active at some stage or not. In my opinion, looking at just the protein levels isn't confirmatory and only suggestive that that caspases aren't involved. All the best.

The protein level of caspases is going down most probably due to postapoptotic degradation. In order to make sure these caspases were not cleaved and activated, you should choose earlier time points, in ideal case a time course should be done.

The level of PARP and cleaved PARP is going down because of the same reason.

I would imagine yes, some cancer cell lines are less sensitive to chemotherapeutic agents because they have down-regulated caspases, but do undergo apoptosis.

I agree with the last observation

Dear Andrew and Joaquin, yes I agree too. But here this is not the case because the untreated cells have normal level of full length caspase and PARP.

Dear Laishram, Here we should clarify that specific cleavege of caspases and degradation is not the same. As well as overexpression and activation is two different phenomena. Caspase cleavage and activation could have happened here but earlier. And the time you harvested the cells might be far too late and the proteins just aspecificaly degraded. As Saurabh suggested an activity assay(for all the possible caspases) would be a very good solution, together with a time course western blot. Only if these tests are negative I would say this cell death is caspase independent.

Laishram, can you elaborate on the morphological characteristics that are apoptotic (i.e. what you are observing?). I agree with the suggestions of using Annexin V/PI. However, this wouldn't entirely exclude the possibility of other forms of non-apoptotic cell death occuring.

Probably, the apoptosis is followed by secondary necrosis, leading to loss of cleaved/activated proteins. You might need additional assays to confirm necrosis, granzyme-B activation.

If cleaved PARP is going down, it is possible that necrosis is taking place and all proteins are being degraded. One can easily check for necrosis by staining for cell integrity-nucleus should be intact and clearly visible. The Annexin V/PI will distinguish between apoptosis and necrosis-but it is more expensive. Perhaps shorter exposure to drug or different batches of drug can be tried, and levels of caspase, PARP etc can be repeated?? One should also repeat with different batch of cells, and maybe use other cervical cancer cell lines--WHY?? because I have seen that different batches of plant extract give diffferent data-and HeLa cells themselves change in culture very often. Hope this helps!

Gavin, I had done AO/EB staining method for detecting morphological characteristics of apoptosis and had observed cells with membrane blebbings, condensed nucleus, fragmented nucleus.

Thank you all for your suggestions. I will try Annexin V/PI and a time course of caspase activity.

Hi Laishram,

You may want to look at the AIF (apoptosis inducing factor), which is released from mitochondria and can induced chromatin condensation and apoptosis independent of caspases. To know more please have a look at the following link which refers to many publications on role of AIF in carcase-independent apoptosis. Hope that helps.

http://scholar.google.ca/scholar?q=caspase+independent+apoptosis&hl=en&as_sdt=0&as_vis=1&oi=scholart&sa=X&ei=PWJ5UY37MMPOiwLto4HABQ&sqi=2&ved=0CCwQgQMwAA

I would suggest RT-PCR just to cut a long story short.

Yes, it can. I found this out in the course of my other research work but i can not say how this happened.

There are a few papers showing apoptosis independent of PARP1 and caspase 3. One way to see would be as Mitra Esfandiarei suggests to measure caspase independent apoptosis-inducing factor (AIF) as during this process it is released from mitochondria. There is a ELISA kit, which would be a easy way to quantitate AIF,. (dont know if I should advertise companies on here so lets just say its a british company with a sheep as their logo). Good luck with your research....

First check in flow cytotoxic by using Annexin  V/PI whether  it is apoptosis  or necrosis. Only in apoptosis you would get signals for Caspase and PARP1. If it is necrosis, there are two ways: accidental death or programmed. Usually in programmed necrosis, known as necroptosis, you get signal for AIF translocation  from mitochondria  to nucleus . It may be related to ATP dropped down and change in mitochondrial  membrane potential. AIF truncation depends on ca the pin or calpin, and translocation rely on HSPD1. Design your experiments to check all these points. Hopefully  you get answer. Best of  luck . 

Thank you Saifur. I thought AIF is related to caspase independent apoptosis and not necrosis. My sample did lower the mitochondrial membrane potential and AnnexinV/PI study in flow cytometry indicated apoptosis.

Caspase independent  apoptosis is basically necroptosis where AIF involved .  Literatures of cell death are so complex and there are no standard classification, though many scientists  and consortium  tried. Every classification has its limitation. Check carefully in flow cytometry and flueresence microscope. Use IC50  concentration with others. Hopefully it would be late apoptosis  dominant (known as necrosis). As this necrosis follow AIF signal and down  stream pathway, it is programmed necrosis, also know as caspase-independent  apoptosis by old fellows who don't  dire to use new term. BUT it is needed to make proper classification. However ,  it's up to you. After all it is your research. Best of luck.

NB. By definition, Apoptosis should be proceed through Caspase signaling  pathway. So, If Caspase is not involved ,  no chance to use the term apoptosis. If you check in microscope, you can see pattern of necrosis there.

Many drugs /plant extracts cause apoptosis at lower concentrations and necrosis at higher concentrations.IC50 can be infuenced by many factors  (exposure time to drug, assay time point, starting cell density..and even the type of assay etc).   It can be useful to comprehensively study these aspects. Does your extract cause any  toxicity to normal cervical epithelial cells? This is another important aspect that would add value to your study. Hope this helps!

Perhaps one of the best t hints you can do is to do a dose response to your extract and measure cell viability.  If the concentration that you are using effectively kills all the cells is is very probable that you are seeing necrosis.  Examine what is happening to your cells at a dose which kills 60 and 90% of the cells and it is more probable that you are seeing apoptosis.  Interesting there are quite a few cell lines in which caspases (for example caspase 3) are mutated or silenced and they play no real role in apoptosis.

good luck! 

Based on my own experience, cleaved PARP is a very sensitive early stage marker for apoptosis. However, when most cells goes apoptosis or necrosis, expression of cleaved PARP will be difficult to detect. In my experiment, I treated cells with anti-cancer drugs and I failed to detected expression of cleaved PARP when around 30% cells were Annexin V+/PI-(early stage apoptosis) and 30% were Annexin V+/PI+(apoptosis and necrosis) in FACS. So I suggest try to low down the concentration of drugs/plant extract or shorten the treatment period. Good luck

Of course they are going down, you are comparing dying or already dead cells with live ones. In the protein lysate from the dying or dead cells, you first of all have lower protein and most of the protein would be degraded. Western Blots are never a good idea in studying cell-death because of this reason. your protein quantification assay would not be able to differentiated between degraded and intact proteins. Once lysosomal enzymes are out, they would destroy every epitope. Moreover, Apoptosis can occur independent of Caspase-3, there are other effector caspases like Caspase-7 for example.

What doees downregulation of caspase 7 says? how would you explain with reference.

Motasim is right. A dying cell is degrading lots of proteins including caspases and other apoptosis related proteins. So, earlier time points and drug doses that cause 60-90% death (as Andrew said) are necessary. HeLa cells are very heterogenous -so its could to check with different passage numbers and cells from another source--Hope this helps!